Complexity of Inflammation in the Trajectory of Vascular Disease: Interleukin 6 and Beyond

Abstract

Takayasu’s arteritis, first described by Dr. Mikito Takayasu in 1908, is a systemic vasculitis that mostly affects the aorta and its major branches. Although the etiology of the disease is yet unknown, genetic and environmental factors may both play a role. One hundred years after the discovery of Takayasu’s arteritis, inflammation is finally widely recognized as a fundamental condition common to all vascular diseases, and clinical trials have proven the efficacy of molecularly targeted drugs that block each step of the NLRP3 inflammasome/interleukin (IL)-1β/IL-6 cascade in patients with atherosclerotic vascular disease and elevated C-reactive protein (CRP). Recent advances have also been made in the treatment of Takayasu’s arteritis. The randomized controlled trials and subsequent open-label and post-marketing surveillance studies in Japan have demonstrated that tocilizumab, an anti-IL-6 receptor antibody, is effective in the treatment of Takayasu’s arteritis and prevents relapse during tapering of prednisolone doses. IL-6 is also heavily engaged in the remodeling of large vessels after acute aortic dissection as demonstrated in animal studies. In patients with acute aortic dissection, those with markedly elevated CRP levels in the acute phase are known to have an increased risk of aorta-related events, such as rupture due to aortic diameter enlargement, in the subacute and chronic phases. We discovered that elevated CRP levels following aortic dissection are caused by IL-6, which is produced by neutrophils that infiltrate the adventitia of the dissected aorta. In a mouse model of acute aortic dissection, we showed that IL-6 produced by these neutrophils causes progressive destruction of the arterial wall structure and that blockade of IL-6 signaling can prevent post-dissection vascular remodeling and improve life outcome. Therefore, inhibiting IL-6 signaling is anticipated to be effective in the secondary prevention of myocardial infarction and suppression of vascular modeling after dissection and even as an anti-inflammatory therapy for Takayasu’s arteritis; however, this approach does not solve everything. Undoubtedly, the mechanisms of inflammation in vascular disease are diverse and complex, and the cytokines and cell populations involved at each site (coronary artery vs. aorta) and in each phenotype (atherosclerosis vs. aortic aneurysm vs. aortic dissection) need to be understood for each type of inflammation. Osteopontin (OPN) is a recruiter of monocytes and macrophages, induces cellular immune responses as a Th1 cytokine, acts as a fibrosis-promoting factor, and has been shown to be deeply involved in the pathogenesis of vascular diseases. We have shown that senescent T cells, which emerge with obesity and aging, secrete significant amounts of OPN, leading to metabolic abnormalities and chronic inflammation. Neutrophil extracellular traps (NETs) released from activated neutrophils have been shown to contribute to the pathogenesis of acute coronary syndromes (ACS) by interacting with macrophages, platelets, and vascular endothelial cells and thus promoting plaque erosion and immunothrombosis. In addition to standard anticoagulant and antiplatelet therapies, the effectiveness of anti-immunothrombotic therapies targeting NETs as a new preventive and therapeutic approach for ACS will be examined in the future.

Atherosclerotic Disease and IL-6 Signaling

One hundred years after the discovery of Takayasu’s arteritis, inflammation is now being considered as the next therapeutic target in cardiovascular disease after the development of lipid-lowering statins, and elevated high-sensitivity C-reactive protein (CRP) has been recognized as an independent predictor of recurrent ischemia and death in patients with coronary artery disease. The JUPITER (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) trial found that in patients with normal low-density lipoprotein cholesterol but high CRP levels, statins reduced the incidence of major cardiovascular events by 79% in the group in which both CRP and low-density lipoprotein cholesterol decreased.¹⁾ In the CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) trial, canakinumab, a fully human anti-interleukin (IL)-1β monoclonal antibody, reduced the rate of recurrent nonfatal myocardial infarction in patients with prior myocardial infarction who had high-sensitivity CRP (hs-CRP) levels of 0.2 mg/dL or higher.²⁾ Interestingly, an exploratory analysis of the CANTOS trial revealed that canakinumab reduced lung cancer incidence and mortality by approximately 75%,³⁾ suggesting that controlling inflammation in the tumor microenvironment may provide additional clinical benefits.

Cholesterol crystals are well known to activate NLRP3 in atherosclerotic foci,⁴⁾ but NLRP3 can be activated also by a number of damage-associated molecular patterns, including uric acid crystals⁵⁾ and calciprotein particles,⁶⁾ which are deposited in the arterial wall in lifestyle-related diseases. Two clinical trials have tested whether suppression of the NLRP3 inflammasome itself with colchicine can reduce the risk of cardiovascular events^7,8): In COLCOT (Colchicine Cardiovascular Outcomes Trial), which was performed in patients soon after they had experienced a myocardial infarction, and LoDoCo2 (Low-Dose Colchicine 2), which was carried out in patients with coronary artery disease whose condition has been stable for at least 6 months, the addition of colchicine 0.5 mg/day to secondary prevention therapy further reduced the risk of cardiovascular events. A proteomic substudy of LoDoCo2 confirmed that colchicine attenuates the NLRP3 inflammasome pathway, including IL-18, IL-1 receptor antagonist, and IL-6, in patients with chronic artery disease.⁹⁾

The findings of these clinical studies demonstrated that inflammation plays a role in the etiology of cardiovascular disease and that anti-inflammatory therapy has clinically significant effects. IL-6 is now attracting attention as a downstream molecule of NLRP3 inflammasome/IL-1β.¹⁰⁾ In the CANTOS trial, patients with a clear reduction in IL-6 after the first dose of canakinumab experienced fewer serious adverse cardiovascular events over the course of long-term treatment. Furthermore, genetic analysis revealed that IL-6 signaling is also associated with the risk of stroke, coronary artery disease, aneurysms, calcific aortic stenosis, and atrial fibrillation.

IL-6 performs its biological function through two types of molecules, IL-6 receptor (IL-6R) and glycoprotein 130 (gp130).¹¹⁾ Binding of IL-6 to membrane-bound IL-6R (mIL-6R) forms a high-affinity receptor complex consisting of IL-6, IL-6R, and gp130. IL-6 can also bind to soluble IL-6R (sIL-6R), which lacks the intracytoplasmic portion of mIL-6R, and the IL-6/sIL-6 complex can also form a complex with gp130. This unique receptor signaling system is called IL-6 trans-signaling.¹²⁾

Patients with chronic kidney disease (CKD) and higher hs-CRP are at high cardiovascular risk. The RESCUE (Reduction in Inflammation in Patients with Advanced Chronic Renal Utilizing Antibody-Mediated IL-6 Inhibition) study, which examined the effect of the investigational once-monthly drug ziltivekimab, an IL-6 monoclonal antibody, in these patients, showed significant reductions in multiple inflammatory biomarkers associated with atherosclerosis.¹³⁾ Consequently, the ongoing cardiovascular outcomes trial ZEUS (Ziltivekimab Cardiovascular Outcome Study) was started to compare ziltivekimab with placebo in roughly 6,000 patients with CKD and elevated hs-CRP and find out whether the new anti-inflammatory strategy of directly reducing IL-6 in circulating blood reduces rates of cardiovascular events. The reasoning behind these trials may well have been that colchicine cannot be used in the presence of renal failure and that, in Western countries, patients with CKD represent a high-risk population for myocardial infarction and stroke. In contrast, hs-CRP is rarely elevated in Japanese patients with atherosclerotic disease, and Japanese patients with CKD have a lower risk of developing cardiovascular disease than patients with CKD in Western countries. Japanese patients with CKD are not at much higher risk of myocardial infarction or stroke than the general population, but they do have a higher risk of heart failure and all-cause mortality.¹⁴⁾ As a central player in the neuro–immune–metabolic interface, IL-6 has been shown in patients with chronic rheumatoid arthritis to be deeply involved not only in the immune system but also in disturbances of the metabolic insulin resistance and lipid metabolism and nervous systems (pain, fatigue, anxiety, depression) that affect patients’ quality of life (Fig. 1). Therefore, despite ethnic disparities in the prevalence of vascular disease and the positive frequency of high-sensitivity CRP, the outcomes of the ongoing analysis of clinical trials with IL-6 antagonists are eagerly anticipated.

Fig. 1 Various actions of interleukin-6 (IL-6) in vascular diseases. IL-6 plays a significant role in the pathogenesis of vascular diseases as a key participant in the neuro–immune–metabolic interface.

Inflammation and IL-6 after Aortic Dissection

In acute aortic dissection, the aortic intima is torn and blood enters the aortic wall through the tear. Acute type A aortic dissection has an extremely poor prognosis and is treated by emergency surgery, i.e., ascending aortic replacement. On the other hand, acute type B aortic dissection has a better spontaneous prognosis, and conservative treatment, mainly antihypertensive therapy, is appropriate in the acute phase. Nevertheless, type B dissection must also be closely watched during the subacute and chronic phases because of the risk of aortic enlargement (aneurysm formation) and subsequent rupture. The size of the increase in CRP levels in the acute phase has been reported to be a useful prognostic factor in acute aortic dissection, and patients with high acute-phase CRP levels are at increased risk of complications, including rupture due to aortic enlargement.¹⁵⁾

CRP levels in the acute phase may reflect the degree of inflammation in the dissected aorta, and higher CRP levels may suggest that fatal complications, including rupture, can be avoided by intervening in inflammation of the arterial wall following dissection. To enable research to be performed on the mechanisms of inflammation in the dissected aorta, a mouse model is needed that allows researchers to predict when dissection will occur. With the traditional method of inducing dissociation by injecting angiotensin II into apoE−/− or aged mice, vascular inflammation could not be monitored over time because dissociation occurred by chance and it was impossible to forecast when it would happen. Fortunately, a research team led by Professor Yasunori Okada of the Department of Pathology at Keio University, Tokyo, Japan, has created a novel mouse model of aortic dissection that uses the lysyl oxidase inhibitor β-aminopropionitrile monofumarate. The enzyme lysyl oxidase is essential for extracellular matrix stabilization, particularly for the enzymatic cross-linking of collagen and elastin, and in mice treated with β-aminopropionitrile monofumarate, the vessel wall is weakened, and administration of angiotensin II causes aortic dissection in 100% of mice within 24 h.¹⁶⁾

We used this mouse model to examine inflammation of the aortic wall over time after aortic dissection.¹⁷⁾ We discovered that expression of C-X-C motif chemokine ligand 1 and granulocyte colony stimulating factor was induced in the adventitia of aortas after dissection and that these neutrophil chemotactic factors directed the infiltration of neutrophils released from the bone marrow. An important finding was that the infiltrating neutrophils produced IL-6, which amplified inflammation in the aorta after dissection, further damaging its wall structure and leading to aneurysm formation and rupture (Fig. 2). In fact, we discovered that suppressing IL-6 signaling greatly increased survival by preventing rupture after aortic dissection. If the results of this study are translated into clinical practice, blocking IL-6 signaling in patients with markedly elevated hs-CRP levels in the acute phase after aortic dissection can be expected to reduce aortic inflammation and prevent aortic-related events such as rupture due to aortic expansion.

Fig. 2 Mechanisms of vascular inflammation after aortic dissection. Neutrophil chemotactic factor CXCL-1/G-CSF is expressed on the adventitia side of the dissected aorta. Neutrophils mobilized from the bone marrow infiltrate the adventitia. Structural destruction of the aortic wall progresses with interleukin-6 (IL-6) produced by neutrophils.

Takayasu’s Arteritis and IL-6

Takayasu’s arteritis is a vasculitis that causes inflammation of the aorta and large vessels branching off the aorta. The three ascending branches from the aortic arch are particularly prone to involvement, but the lesions also extend into the descending aorta. The ascending aorta becomes dilated, resulting in ascending aortic aneurysm and aortic regurgitation. Clinically, persistent chronic inflammation of these vessels causes systemic symptoms, such as fever and general malaise, and various vascular symptoms due to vasodilation, stenosis, and occlusion. Histologically, during the active phase of inflammation, a strong inflammatory cell infiltrate spreads from the adventitia to the tunica media, with a worm-eaten loss of elastic fibers in the tunica media layer. In the scar stage, the tunica media thins, the adventitia shows fibrous thickening, and the intima also shows diffuse thickening with plate-like calcification. The age of onset of the disease is between 10 and 30 years, with a peak in the 20s, and the condition occurs more frequently in women (the male-to-female ratio is approximately 1 : 9). Traditionally, corticosteroids such as prednisolone (PSL) and immunosuppressive drugs have been used to treat Takayasu’s arteritis, but in the past few years, three clinical studies^18–20) and one post-market surveillance study²¹⁾ from Japan have evaluated the efficacy of tocilizumab, a humanized antihuman IL-6 receptor monoclonal antibody.

The first study was a randomized, double-blind, placebo-controlled, phase III trial (TAKT study) in patients with refractory Takayasu’s arteritis who had relapsed on PSL 0.2 mg/kg/day.¹⁸⁾ Initially, remission was achieved by doubling the PSL dose; then, PSL was forcefully tapered off at a defined rate. The primary endpoint was time to first relapse of Takayasu’s arteritis. Thirty-six patients were enrolled in the study and randomized 1 : 1 to placebo or tocilizumab. The data indicated a tendency for tocilizumab to reduce recurrence, although the primary endpoint did not approach statistical significance (hazard ratio, 41%; 95% confidence interval, 0.15–1.10; p=0.0596). After completion of the randomized controlled study, all 36 patients were administered tocilizumab for 96 weeks and followed; 28 subjects completed the open-label research. In the open-label study, the administration of corticosteroids was left to the discretion of the attending physician.¹⁹⁾ Since the TAKT study included patients who relapsed even on PSL 0.2 mg/kg/day, the mean PSL dose at relapse prior to entry into the study was 0.223 mg/kg/day. The PSL dose was doubled to achieve remission in all patients, so the PSL dose at the time of randomization was 0.439 mg/kg/day. At the end of the subsequent open-label observational study at 96 weeks, the PSL dose had been reduced to 0.105 mg/kg/day (<0.2 mg/kg/day in 85.7% of patients and <0.1 mg/kg/day in 46.4% of patients). However, during the open-label follow-up study, 18 relapses were recorded in 14 patients.

In a post hoc analysis of the TAKT research, changes in vascular lesions before and after tocilizumab treatment were investigated retrospectively by analyzing computed tomography (CT) images of 28 patients for whom follow-up imaging results were available. In both the tocilizumab and placebo groups, wall thickening, stenosis (occlusion), enlargement (aneurysm), and contrast effects of 22 vessel segments were compared before double-blind treatment and at the end of the 96-week open-label tocilizumab administration.²⁰⁾ Among the 28 patients, a total of 57.1% displayed improved or maintained wall thickening; 10.7%, partial progression (worse than baseline) in one or more vessels; and 28.6%, new progression (baseline findings were normal, but wall thickening was observed at 96 weeks).

A few other cases of vascular complications such as thickening and enlargement of the vessel wall (aneurysms) during gradual PSL reduction with tocilizumab administration have been reported.^22,23) Inflammatory indicators such as CRP are ineffective for monitoring disease activity when taking tocilizumab. Therefore, it is crucial to carefully monitor the development of new wall thickening by contrast-enhanced CT when tocilizumab is administered and PSL is tapered. Progression or new development of wall thickening clearly indicates the need to consider intensifying treatment, such as increasing the PSL dose.

Because the sample size in the TAKT study was small, Chugai Pharmaceutical Co., Ltd. evaluated the safety of the tocilizumab in a post-marketing surveillance study with a target of 100 patients.²¹⁾ In this study, 120 patients (95% female; mean age, 38.4 years; mean age of onset, 30.3 years) were assessed for safety, changes in PSL dosage, improvement in clinical symptoms, and recurrence rate. Tocilizumab was prescribed as the initial treatment in 46.7% of patients and as the treatment for relapse in 50.8%. Serious infections occurred in 7.5% of the patients. A total of 96 (80.0%) of the patients did not relapse, and in 83.0% (n=80) of these patients, the concomitant dose of corticosteroids could be reduced to less than 10 mg/day (PSL equivalents). However, 24 (20%) of the patients relapsed over the 52-week observation period. These results reaffirm the importance of carefully reducing the dose of steroids, even when they are used in combination with tocilizumab, with the goal of achieving the lowest dose that does not cause vascular complications.

Vascular Disease and Osteopontin

In addition to the components of the NLRP3 inflammasome/IL-1β/IL-6 pathway, several other molecules, including osteopontin (OPN), have drawn interest as potential therapeutic targets in vascular system inflammation.^24,25) OPN was first discovered as a glycoprotein in bone matrix. Subsequently, it has been demonstrated to be crucially involved in chronic inflammation, organ remodeling, autoimmune diseases, and cancer invasion and metastasis as a molecule involved in monocyte and macrophage recruitment as a pro-inflammatory cytokine, induction of cellular immune responses as a Th1 cytokine, and extracellular matrix remodeling and fibrosis. In humans, hyperosteopontinemia is a poor prognostic predictor of obesity, diabetes, and heart failure. On the other hand, studies in centenarians have reported that hyposteopontinemia is a marker of “successful aging.” In the cardiovascular system, OPN is rarely expressed in the normal physiological state, but its expression is markedly increased in pathological conditions.²⁶⁾ It is instantly produced after injury and serves a crucial part in wound healing, but it swiftly fades once healing is complete. On the other hand, in situations in which OPN expression is persistently increased, a chronic inflammatory state develops, promoting the destruction of tissue structures.²⁷⁾

Studies have revealed that persistently elevated serum OPN levels are a predictor of serious adverse cardiovascular events^28,29) and that OPN is expressed in human atherosclerotic plaques.³⁰⁾ In patients who have undergone carotid endarterectomy, higher OPN expression in plaques is associated with the development of unstable plaques.³¹⁾ Patients with acute coronary syndromes have higher serum levels of OPN than those with stable coronary artery disease. Increased serum OPN levels are linked to rapidly progressing coronary plaque and in-stent restenosis.³²⁾ In mice, overexpression of OPN accelerates the progression of atherosclerosis, and conversely, knockout of OPN suppresses atherosclerosis progression.^33,34) In humans, OPN is also known to prevent ectopic calcification, such as atherosclerotic plaque and aortic valve calcification.^35–37)

The function of OPN in the process of atherosclerotic plaque formation, growth, and failure is complex, and generation of various isoforms through alternative splicing or posttranslational modifications such as phosphorylation may contribute to the complexity of OPN function.³⁸⁾ In obesity and CKD, overproduction and overflow of OPN may occur in nonvascular organs and secondarily intensify chronic inflammation of blood vessels throughout the body. Obesity is often associated with insulin resistance, type 2 diabetes, dyslipidemia, and hypertension, and the term metabolic syndrome is used to refer to the condition in which several of these risk factors for atherosclerosis are combined. In addition to contributing to atherosclerosis, metabolic syndrome is associated with a number of other health problems, including nonalcoholic fatty liver disease, dementia, and airway disease, and it also increases the risk of several cancers. The state of chronic low-grade inflammation associated with excess adipose tissue was shown to explain the development of obesity-related conditions, and research has suggested that in obese patients, elevated serum OPN levels are associated with metabolic syndrome and may be useful as a diagnostic biomarker for this condition. We have shown that age-associated T cells (CD153⁺PD-1^hiCD44^hiCD4⁺T cells), which are normally absent in healthy nonobese young mice but appear in aged mice, rapidly appear in visceral adipose tissue and secondary lymphoid tissues such as the spleen when obesity is induced by high fat diet load (Fig. 3). This T lymphocyte population acquires an aging phenotype and produces high levels of OPN as a senescence-associated secretory phenotype that increases the content of OPN in the blood. We further demonstrated that OPN produced by this T lymphocyte population plays an essential role in the chronicity of visceral fat inflammation.^39,40) OPN secreted by senescent T cells may operate as an initiator of metabo-aging diseases linked to aging and obesity. The so-called senolytic therapy, which selectively eliminates (controls) senescent T cells, is expected to be useful in antiaging medicine for metabolic syndrome patients and the elderly.

Fig. 3 Cellular senescence of T cells is a common mechanism involved in obesity-associated and aging-associated diseases. The inflammatory response in which aging T cells produce osteopontin (OPN) has no brakes at all, i.e., the immune checkpoint mechanism is disrupted. Aged T cells express high levels of PD-1 in their cellular expression. Adipocytes, macrophages, and B cells, the major antigen-presenting cells in obese visceral fat, strongly express PD-L1. Secretion of OPN from CD153⁺PD-1^hi CD4 T cells is not inhibited by PD-1-mediated signals.

Recent studies have conclusively established a link between CKD and cardiovascular disease, including myocardial infarction, angina pectoris, heart failure, and stroke. Cardiorenal syndrome originally emerged as a term to characterize the bidirectional adverse effects of reduced cardiac function on renal function and vice versa.⁴¹⁾ However, recently there has been renewed interest in the involvement of cardiorenal syndrome in the pathogenesis of both cardiovascular and renal diseases in diabetes. The hypothesis is that effects of diabetes on the kidneys lead to cardiovascular injury via activation of neurohumoral factors. OPN is recognized as one of the key molecules causing organ damage in diabetes-associated cardiorenal syndrome.⁴²⁾ Diabetic kidney disease (DKD) begins with glomerular vascular endothelial injury and glomerular hyperfiltration due to metabolic abnormalities associated with type 2 diabetes, followed by the appearance of microalbuminuria. Subsequently, as podocyte damage progresses to overt proteinuria, the tubules are exposed to proteins, causing tubular damage and loss of nephrons and eventually leading to end-stage renal failure. Consequently, glomerular injury has long been thought to be the cause of DKD that manifests as albuminuria. However, sodium-glucose cotransporter-2 (SGLT2) inhibitors, which act on proximal tubular epithelial cells, have been shown to significantly improve the prognosis of DKD, a finding that highlights the importance of metabolic stress in the proximal tubules in the pathogenesis of DKD. SGLT2 inhibitors protect the kidneys of patients with diabetes and break the vicious cycle of the cardiorenal cycle, thus delaying the onset of cardiovascular disease.^43–45) After being filtered by the glomerulus, 100% of glucose is reabsorbed in the proximal tubules; in people with diabetes, a larger amount of glucose is reabsorbed in the proximal tubules than in nondiabetic individuals. In addition, about 66% of sodium filtered through the glomerulus is reabsorbed in the proximal tubules, and this percentage is also higher in diabetes. These glucose and sodium reabsorptions are coupled with Na⁺/K⁺ ATPase activity.⁴⁶⁾ In diabetes, oxygen consumption in the proximal tubules is increased, and the partial pressure of oxygen in the surrounding tissues is decreased. A recent metabolome/metabolic flux research revealed that metabolic reprogramming occurs in the proximal tubular epithelial cells in type 2 diabetes, resulting in increased metabolic flux in the glycolytic pathway.⁴⁷⁾ Abnormal glycolytic pathways are detrimental to proximal tubular epithelial cells. SGLT2 inhibitors suppress the abnormal glycolysis in diabetic kidneys.⁴⁸⁾ In cultured proximal tubular epithelial cells, we discovered that a high glucose environment encourages the glycolytic pathway and boosts the creation of mitochondrial reactive oxygen species (ROS) and the transcription of OPN. Inhibition of the glycolytic pathway or inhibition of glucose uptake into the cell by SGLT2 inhibitors can suppress mitochondrial ROS generation and OPN transcriptional activity.⁴⁹⁾ Thus, in diabetic kidney disease, OPN is a helpful indicator for proximal tubular overload. It is quite possible that OPN produced from the proximal tubules not only causes renal fibrosis but also acts systemically to accelerate cardiovascular inflammation and remodeling.

Vascular Disease and Neutrophil Extracellular Traps (NETs)

NETs actively spread their own nuclear deoxyribonucleic acid in a mesh-like pattern around their periphery, trapping pathogenic microorganisms such as bacteria and viruses, and at the same time, they release antimicrobial proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G, and histones that attach to the DNA, thereby preventing infection. The formation of NETs was discovered in 2004 as a new mechanism of innate immunity.⁵⁰⁾

NET formation typically requires the generation of ROS. ROS-dependent activation of protein arginine deiminase 4 (PAD4) promotes histone citrullination, nuclear membrane loss, and release of NET components.⁵¹⁾ Hyperglycemia encourages NET synthesis by activating nicotinamide adenine dinucleotide phosphate oxidase and increasing ROS production.^52,53) NET development is impaired in NE knockout mice and MPO-deficient patients, proving that NE and MPO are both necessary for NET formation.^54,55) Activation of the NLRP3 inflammasome in macrophages causes NET formation via production of IL-1β and IL-18.⁵⁶⁾ Conversely, NETs activate the NLRP3 inflammasome in macrophages. Histones produced from NETs directly interact with toll-like receptor 2 (TLR2) expressed on T cells, inducing differentiation into T helper 17 cells, which in turn mobilize a significant amount of neutrophils.⁵⁷⁾ In this way, NETs amplify neutrophil-centered inflammation.

There is growing evidence that neutrophil–macrophage communication sparks an inflammatory response that worsens atherosclerosis and that NETs are involved in this response.⁵⁸⁾ Cholesterol crystals cause macrophages to produce pro-IL-1β, and they activate inflammasomes to facilitate conversion of pro-IL-1β into active IL-1β. Cholesterol crystals also cause neutrophils to generate NETs, which work in concert with cholesterol crystals to produce IL-1β in macrophages. Secreted IL-1β stimulates IL-17 production from T cells and amplifies neutrophil recruitment to plaques.⁵⁸⁾

Recently, the development and popularization of intracavitary imaging revealed that erosion of plaques that retain their fibrous capsules accounts for one-third of cases of acute coronary syndrome (ACS).⁵⁹⁾ There is growing evidence that NETs contribute to the degradation of the fibrous capsules of plaques (Fig. 4). Alterations in the shear stress gradient activate TLR2 on endothelial cells, causing loss of basement membrane integrity and shedding of endothelial cells. Plaque erosion results from direct contact between flowing blood and the endothelial surface, which is devoid of endothelial cells, and involves NET formation.^60,61) The interaction of neutrophils and thrombin-activated platelets at the injured lesion further promotes NET formation, ultimately inducing the formation of an immune thrombus.⁶²⁾ Thus, NETs may be crucial to the emergence of ACS. In addition to anticoagulation and antiplatelet therapy, it would be of interest to evaluate whether anti-immunothrombotic therapies targeting neutrophils are effective in preventing or treating ACS.

Fig. 4 Neutrophil extracellular traps (NETs) play an important role in the development of ACS. Not only monocytes and macrophages, but also neutrophils, are important in the pathogenesis of vascular disease. Neutrophils are primed by various stimuli, including chemokines, cytokines, and damage-related molecular patterns. The activated neutrophils contribute to the vascular inflammation through degranulation, phagocytosis, reactive oxygen species generation, and release of NETs. In particular, NETs promote immunothrombosis through interaction with vascular endothelial cells and platelets and are implicated in the development of acute coronary syndrome, deep vein thrombosis, and COVID-19-associated coagulopathy.

DNA: deoxyribonucleic acid; NE: neutrophil elastase; MPO: myeloperoxidase

At present, PAD4 blockade and DNase I-driven removal are the center of study as possible treatments for NETs. In addition, we recently found that hydrogen gas (H₂) suppresses NET production by activated neutrophils and ameliorates inflammatory responses. H₂ can remove potent oxidizing radicals, such as hydroxyl radicals, and has demonstrated therapeutic effects in a number of disorders involving ischemia–reperfusion injury.^63–65) To obtain regulatory approval for H₂ as a medical gas, we performed the HYBRID II (Efficacy of Inhaled Hydrogen on Neurological Outcome Following Brain Ischemia During Post-Cardiac Arrest Care) trial and showed that H₂ inhalation may improve life expectancy and neurological outcome in patients on temperature management therapy after cardiopulmonary resuscitation; we presented our findings at the Resuscitation Science Symposium 2022 (November 5–6, 2022, Chicago, IL, USA).

In addition, a study in China revealed that H₂ inhalation therapy is successful in avoiding severe cases of COVID-19 pneumonia.⁶⁶⁾ Microvascular thrombosis caused by NETs has been implicated in the severity of COVID-19 pneumonia, so we hypothesized that inhaled H₂ may reduce the severity of COVID-19 pneumonia by inhibiting NET formation in the pulmonary vasculature. In a study designed to test this theory, we discovered that H₂ inhibited both ROS-dependent NET formation by phorbol myristate acetate and ROS-independent NET formation by the calcium ionophore in vitro. Furthermore, in a porcine model of lipopolysaccharide-induced sepsis, we confirmed that H₂ inhalation inhibited NET formation in the circulating pulmonary blood.⁶⁷⁾

H₂ can be administered to patients at the bedside through a H₂ generator or gas cylinder. It has also been shown to increase the integrity of vascular endothelial function and stabilize hemodynamics during hemorrhagic shock.⁶⁸⁾ Thus, H₂ has the potential to be widely utilized in the treatment of vascular diseases.

Conclusion

One hundred years after the discovery of Takayasu’s arteritis by Dr. Mikito Takayasu, the complex mechanisms of inflammation in the development of cardiovascular disease are now being clarified. In fact, molecularly targeted drugs that block each step of the pro-inflammatory NLRP3 inflammasome/IL-1β/IL-6 pathway have been developed and show clinical efficacy against atherosclerotic cardiovascular diseases. However, these drugs do not control all vascular inflammation because there is no single mechanism of inflammation in atherosclerosis, aortic aneurysms, aortic dissection, and aortic valve degeneration. Vascular inflammation can be caused by the immune abnormalities seen in Takayasu’s disease; infections, as recently highlighted by COVID-19; vascular responses to damage-associated molecular patterns and shear stress; cellular senescence of vessel wall component cells; and systemic chronic inflammation associated with metabolic syndrome and CKD. Therapeutic attempts to protect blood vessels by understanding and controlling the mechanisms of vascular inflammation are just beginning.

Funding

This research did not receive any specific grant from funding agencies in the public, private, or nonprofit sectors.

Declaration of Interests

I have received lecture fees from the following companies: AstraZeneca K.K., Ono Pharmaceutical Co. Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Pharma Ltd., Novartis Pharma K.K., Mochida Pharmaceutical Co., Ltd., Otsuka Pharmaceutical, and MSD K.K. I have received research funding from the following companies: Nippon Boehringer Ingelheim Co. Ltd., Taiyo Nippon Sanso, Doctors Man, and Ito En.

I have a relationship with Taiyo Nippon Sanso and Doctors Man Co. Ltd., Japan, that includes consulting or advisory.

I am the registered inventors of the patent jointly filed by Keio University and Taiyo Nippon Sanso and Keio University and Doctors Man.

Japanese College of Angiology bestowed the 2022 Takayasu Mikito Prize on the author. This is an invited review article of the achievement.