key: cord-0048176-8zyse31l
authors: nan
title: Full Issue PDF
date: 2020-07-27
journal: JACC Basic Transl Sci
DOI: 10.1016/s2452-302x(20)30296-5
sha: 0dc559bf5ba9118edb13e6c4fa71380ad0d92c53
doc_id: 48176
cord_uid: 8zyse31l

nan

Aortic stenosis was less prevalent in carriers of the PCSK9 R46L variant.

Variation at the PCSK9 locus influences LDL-C levels, but not Lp(a).

PCSK9 is produced and secreted by aortic valves.

In vitro, PCSK9 inhibition might lower calcification in aortic valve cells.

PCSK9 inhibition could represent a therapeutic strategy for aortic stenosis.

The authors investigated whether PCSK9 inhibition could represent a therapeutic strategy in calcific aortic valve stenosis (CAVS). A meta-analysis of 10 studies was performed to determine the impact of the PCSK9 R46L variant on CAVS, and the authors found that CAVS was less prevalent in carriers of this variant (odds ratio: 0. (2) (3) (4) or decrease CAVS incidence (5) . Coronary artery disease (CAD) and CAVS share many risk factors and pathophysiological mechanisms (6) .

Whether other cardiovascular drugs could be effective for the treatment of CAVS is unknown.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme secreted by the liver that binds to the LDL receptor (LDLR) and targets it for lysosomal degradation (7) . Genetic association studies have shown that natural variations at the PCSK9 locus (present in 2% to 3% of the population) are associated with lifelong exposure to low LDL-C levels, and cardiovascular protection (8, 9) . PCSK9 inhibitors have been shown to substantially lower LDL-C levels in various populations and reduce the risk of adverse cardiovascular outcomes in patients at high cardiovascular risk (10, 11) .

Lifelong low LDL-C levels has also been linked to lower aortic valve calcium accumulation and protection against CAVS (12) . Recently, investigators of the Copenhagen General Population Study, the Copenhagen City Heart Study, and the Copenhagen Ischemic Heart Disease Study observed that individuals carrying the R46L variant in PCSK9 are characterized by lower levels of LDL-C, lipoprotein(a) (Lp(a)), and a lower risk of CAVS (13) . However, these results have not been replicated, and it is still unclear whether the cardiovascular benefits are due to changes in LDL-C, Lp(a), or both. It also remains unclear whether PCSK9 is expressed in human aortic valves and whether it contributes to valve interstitial cell (VIC) calcification.

The objectives of our study were to determine whether variation at the PCSK9 locus are associated with plasma lipoprotein-lipid levels and CAVS. We also sought to determine which parameter(s) of the lipoprotein-lipid profile (LDL-C and or Lp[a]) best explained the potential benefits of genetically mediated PCSK9 inhibition for CAVS prevention. We also evaluated whether PCSK9 was present in the aortic valves, whether isolated human VICs could secrete PCSK9 under pro-osteogenic conditions, and whether blocking PCSK9 could mitigate the impact of pro-osteogenic conditions on human VIC calcification.

VARIANT AND CAVS. We investigated the association between the R46L PCSK9 variant and CAVS in a control patients, the QUEBEC-CAVS study 1,009 cases and 11,625 control patients, and 3 French cohorts 3,123 cases and 6,532 control patients). This metaanalysis was performed after each study had tested the impact of this variant on CAVS using logistic regression adjusted for age and sex, and the first 10 ancestry-based principal components when available.

We performed a fixed-effect meta-analysis using the inverse-variance weighted method as implemented in the rmeta package (version 3.0) in R version 3. Table 1 .

EPIC-NORFOLK STUDY. We selected independent (in low linkage disequilibrium) single nucleotide polymorphisms (SNPs) (r 2 < 0.10) at the PCSK9 locus (within 100 Kb of the gene) associated with LDL-C levels at a genome-wide level of significance in the Global Lipids Genetics Consortium (GLGC) (14) . This approach yielded 11 SNPs independently associated with LDL-C levels. Of these, 10 were successfully genotyped in the EPIC-Norfolk study (described in Supplemental Table 2 ). The design of the EPIC-Norfolk prospective population study has been published previously (15, 16) . We built a weighted genetic risk score (wGRS) using these 10 SNPs weighted by the effect of each SNP on LDL-C levels in the GLGC.

We Isolation of aortic VICs was performed using a modified method described by Poggio et al. (19) .

Briefly, aortic leaflets were placed in 2 mg/ml type II collagenase (Worthington Biochemical Corporation, Figure 4C ). Finally, inhibition of extracellular PCSK9 was able to significantly reduce the calcium deposition both in normal medium (À6.3%

[IQR: À6.8% to À0.4%]; p ¼ 0.02) and in osteogenic medium (À56.5% [IQR: À63.3% to À40.0%];

p < 0.0001) compared with their respective control medium ( Figure 4D ).

Previous genetic association studies observed that carriers of genetic variants in PCSK9 associated with low LDL-C levels are at lower risk of a broad range of atherosclerotic cardiovascular diseases (20) . In this study, we confirmed that variants in PCSK9 associated with lower LDL-C levels are also associated with a lower risk of CAVS, and for the first time to our knowledge, we demonstrated that PCSK9 is present in human aortic valves. Investigating the potential parameters of the lipoprotein-lipid profile that might explain the benefits of carrying these variants, we

found LDL-C, but not Lp(a), levels to be a key factor that may explain the benefits of carrying PCSK9 variants for CAVS prevention. The potential benefits of targeting PCSK9 for CAVS prevention were also supported by our in vitro experimental approach,

showing that treating VICs with a neutralizing PCSK9

antibody reduced calcium accumulation in VICs under pro-osteogenic stimuli.

In a previous report of 2,373 individuals included in a nested case-control design of the EPIC-Norfolk study, we have shown that carriers of the PCSK9 R46L variant had lower levels of nuclear magnetic resonance spectroscopy-measured VLDL and LDL particle concentrations and lower Lp(a) (21) . In another study, Sliz et al. (22) reported no impact of this variant on VLDL lipid measures. In our study, we found a significant association between PCSK9 variants and CAVS in the UK Biobank. However,

given the relatively small number of CAVS cases in the UK Biobank (1, 350) , these results are not definitive, and additional studies are needed to confirm these findings and to determine whether the impact of reduced PCSK9 function is independent of the presence of concomitant CAD because approximately one-half of the patients with CAVS also have CAD. Our results confirm those of the Copenhagen cohorts (13) and extend our previous observations that Pcsk9 À/À mice are less likely to develop aortic valve calcification than wild-type mice (23) . Results of our previous study, confirmed by the present one, also suggest that there is a significant correlation between the amount of PCSK9 produced by VICs and the extent of VIC calcification (23) . Our results are also in line with a report from the CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) consortium and the MDCS study (Malmo Diet and with lifelong LDL-C reduction is associated with lower aortic valve calcium accumulation and lower CAVS incidence (12) .

STUDY LIMITATIONS. In the current study, we included additional participants and PCSK9 variants, (11). Table 4 ). Following the induction of MI, high-resolution ultrasound showed that hearts from DdblGATA Figure 4F ).

Toor et al. percentiles are shown. *p < 0.05. Abbreviations as in Figure 2 . Table 6 ). By 7 days after MI, scar size in tissues tended to be increased in anti-Siglec-F antiserum-treated mice ( Figure  5C ) (p ¼ 0.074), and this was accompanied by adverse remodeling of the left ventricle. High-resolution ultrasound showed that hearts from anti-Siglec-F antiserum-treated mice were more dilated ( Figure 5D) and had greater impairment of left ventricular function than did pre-immune serum-treated mice ( Figure 5E and Supplemental Table 6 ).

Thus, pharmacological depletion of eosinophils in C57BL/6 mice, that have a distinct inflammation and repair phenotype compared with BALB/c mice (14) , To confirm that loss of macrophage polarization during infarct repair was due to eosinophil deficiency, BMD eosinophils were adoptively transferred by intraperitoneal injection into DdblGATA mice before and after MI ( Figure 7C ). Adoptive transfer of BMD eosinophils resulted in restoration of Table 8 ).

Blood eosinophil count is reduced in the acute period following MI, and we have previously shown that low Abbreviations as in Figure 2 .

Toor et al. indicating a switch from a homeostatic to an activated phenotype (13, 20) . Overall, eosinophil recruitment appeared to be low in the heart relative to other Inflammation is essential for infarct repair but when excessive or prolonged is associated with adverse cardiac remodeling (29) . In other settings, eosinophil- Figure 7 . 

eosinophils were often located within epicardial regions and in adjacent pericardial adipose tissue.

Based on a recent study that suggested that pericardial cavity macrophages might directly invade the myocardium following MI (5), the investigators suggested that pericardial adipose tissue could represent a potential source of these cells. Although this represents one possibility, future work will be required to delineate whether eosinophils originate from the bone marrow, spleen, and/or adjacent structures (e.g., pericardial adipose tissue).

Using 

Restenosis limits the efficacy of PTA or stent treatment of atherosclerosis in peripheral and coronary artery disease.

Paclitaxel DCBs effectively reduce restenosis; however, recently, their overall safety profile has been called into question.

In an in vivo molecular-structural imaging study of 25 rabbits with experimental atherosclerosis, DCB-PTA, plain PTA, or sham-PTA was investigated using serial intravascular NIRF-OCT and IVUS.

DCB-PTA reduced lesion inflammation on NIRF-OCT and halted lesion progression on IVUS, compared with PTA or sham-PTA.

These findings indicated the potential for DCBs to serve effectively and safely as a regional anti-atherosclerosis therapy.

Paclitaxel drug-coated balloons (DCBs) reduce restenosis, but their overall safety has recently raised concerns.

This study hypothesized that DCBs could lessen inflammation and reduce plaque progression. Using 25 rabbits Figure 1 shows the overview of the in vivo serial imaging study of rabbit atherosclerosis progression and DCB intervention or control subjects.

Lesions were generated in rabbit aortas using balloon injury in cholesterol-fed animals ( Figure 1 ) as previously performed (13, 14) . After baseline, 6-week survival intravascular NIRF-OCT, and IVUS imaging, Paclitaxel-DCB Angioplasty Suppresses Inflammatory Plaque Progression was performed, as previously described (17) . Quantitative NIRF data were generated using co-registered OCT images to correct the NIRF signal according to the distance between the catheter and the lumen surface, and expressed as both the NIRF concentra- Continuous variables were reported mean AE SD, Table 1 ). derived from generalized linear regression models with 95% confidence intervals. *Adjusted for baseline cholesterol, C-reactive protein, and minimal lumen area, and corresponding atheroma parameter at baseline (6 weeks). †p < 0.001; ‡p < 0.01.

Abbreviations as in Table 1 . as well as the ability of DCBs to serve as a regional endovascular therapy for atherosclerosis.

Inflammation drives atherosclerosis progression (18) , and antiÀinterleukin-1b mediated suppression of inflammation can reduce cardiovascular events in patients (19) . The ability to image inflammation in coronary and peripheral arteryÀsized vessels using high-resolution intravascular NIRF molecular imaging offers a translational approach to understanding the mechanisms of clinical atherosclerosis progression and impaired stent healing (11, 12, 27, 28 

A mitochondrial-derived peptide therapy, HNG, was safe and was delivered as adjunctive therapy with standard-of-care reperfusion in a translational large animal model of myocardial ischemia/reperfusion injury.

HNG reduced infarct size per area-at-risk by 41% with an ischemic time of 60 min followed by 48 h of reperfusion.

The infarct-sparing effects of HNG were abolished when the ischemic time was increased to 75 min followed by 48 h of reperfusion.

The use of rigorous translational large animal models that account for clinically relevant variables is a prerequisite to better predict the clinical efficacy and outcomes of novel therapeutic strategies. 

With the complexities that surround myocardial ischemia/reperfusion (MI/R) injury, therapies adjunctive to reperfusion that elicit beneficial pleiotropic effects and do not overlap with standard of care are necessary. This study found that the mitochondrial-derived peptide S14G-humanin (HNG) (2 mg/kg), an analogue of humanin, reduced infarct size in a large animal model of MI/R. However, when ischemic time was increased, the infarctsparing effects were abolished with the same dose of HNG. Thus, although the 60-min MI/R study showed that HNG cardioprotection translates beyond small animal models, further studies are needed to optimize HNG therapy for longer, more patient-relevant periods of cardiac ischemia. This MDP is widely conserved across species and is an intracellular and endocrine-signaling molecule (11, 12) . Humanin was initially shown to be cytoprotective through interaction with insulin-like growth factor-binding protein-3 (IGFBP3) (13) and inhibition of Bcl-2-associated X protein (Bax) activity (11) . Iko- Humanin has been shown to be a key regulator in mitochondrial homeostasis and other regulatory pathways within the cell that confer cytoprotection, specifically to stress in multiple organs (14) (15) (16) (17) (18) (19) (20) (21) (22) .

Humanin is an intracellular signaling peptide that is also found in circulation, suggesting that it may also possess endocrine properties. Due to the small size of humanin, a thorough investigation into the functional characteristics of each amino acid has been performed (14) . It was determined that the point mutation at serine 14 to glycine (i.e., S14G-humanin (Figures 2B and 2D) . These data indicate the Values are mean AE SD. The p value is between groups at time point for given measurement.

HNG ¼ S14G-humanin. Figure 6A) . Moreover, there was no significant difference (p ¼ 0.837) in the area under the curve as shown in Figure 6B . As shown in Figures 6C and 6D , h-FABP in circulation was not statistically different between groups throughout the study protocol. Figure 8B) . These data suggest that inhibition of 

The kidneys might play a crucial role in regulating systemic NEP actions based on 20 to 100 higher NEP content and activity of the kidneys compared with any other organ.

Tissue NEP expression seems to be downregulated and translates into reduced tissue protein concentrations and activity in HF.

Neither plasma or liquor NEP concentrations and activities reflect tissue NEP regulation; therefore, using NEP as a circulating biomarker seems to be questionable. NEP is a membrane-bound, zinc-dependent metallo-endopeptidase with a wide tissue expression that has high concentrations in kidney tissue, adipose tissue, and lungs (5, 6) . The presence of a nonmembrane-associated or soluble form has also been reported in blood, urine, and cerebrospinal fluid (7, 8) . NEP in the blood stream apparently retains its catalytic activity, which is supported by several studies that reported NEP activity in plasma samples (7) (8) (9) (10) .

Alterations of circulating NEP concentrations and NEP activity have been found to be associated with various diseases, such as metabolic syndrome, lung disease, chronic rheumatoid disease, and Alzheimer's disease (11) (12) (13) (14) (15) (16) . In HF, plasma concentrations of NEP were found to be a risk factor for cardiovascular death in stable patients (17) and were likewise adversely associated with outcome in acute HF (18) . In contrast, no association with outcome was proven for patients with HF and preserved ejection fraction (19) . One study reported measurable circulating plasma NEP activity determined by a fluorometric method, with modest correlation between plasma NEP concentration and activity in stable HFrEF (20) . However, in a mixed cohort of patients with stable and acute HF, plasma Table 1 . Representative images of the CMRIþLE, as well as histology of the ischemia-affected region and control animals are shown in Figure 1A ; neurohumoral biomarkers and myocardial natriuretic peptide expression and content are displayed in Figures 1B and 1C . At the 6-month follow-up, animals with HF had a higher LV enddiastolic volume ( 53.0%; Q1 to Q3: 51.8% to 55.0%; p ¼ 0.008) compared with the control group. Both the peak ejection rate and peak filling rate decreased in the HF animals.

Plasma ANP, renin, and creatinine levels were Corresponding numerical values are listed in Table 2 .

NEP expression was detected in all investigated organs. However, myocardium, liver, and brain samples showed much lower expression compared with the lungs, duodenum, and kidneys. Kidneys showed the highest NEP expression values, which vein. These results were found together with an increased but variable expression of NEP in LV myocardial tissue compared with that in healthy individuals (27) . Our study confirmed detectable NEP expression in cardiac tissues with regional differences with similar expression levels in both ventricles, including the infarcted areas, but found much lower expression in the LA and particularly the RA.

However, in contrast to the previously described findings, our results showed no upregulation but rather unchanged expression in ischemic HF. These Although not approved at the time as a treatment for COVID-19, it could be legally used "off-label," pro- Growing evidence supports the notion that innate immunity is closely related to the development of several CVDs, particularly when the heart responds to ischemia or mechanical stress (10) (11) (12) (13) (14) (15) inflammasome priming and activation signaling. Appropriate NLRP3 inflammasome activation requires an initial priming step triggered by several inflammatory cytokines, which, in turn, upregulates the transcription of the different inflammasome components. Next, a plethora of stimuli ultimately causing potassium (K þ ) efflux induce inflammasome assembly, which is then capable of activating interleukin (IL)-1b and IL-18 as well as gasdermin D (GSDMD), leading to pyroptosis and exacerbating the inflammatory response. (C) Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) signaling pathway. NOD1 agonists mainly consist of pathogen-associated molecular patterns (PAMPs) and/or danger-associated molecular patterns (DAMPs) that induce conformational changes on NOD1, leading to receptor self-oligomerization and recruitment of Receptor-interacting serine/threonine-protein kinase 2 (RIPK2). This adaptor protein then triggers a signaling cascade that subsequently activates NF-kB and MAPKs, eventually upregulating the transcription of pro-inflammatory genes. CVD ¼ cardiovascular disease.

Jaén et al. which creates an atheromatous plaque or atheroma (33) . Atheromatous plaques may progress into unstable plaques, which are characterized by a highly inflammatory and necrotic core infiltrated with macrophages and lymphocytes and a thin fibrous cap that renders these plaques prone to rupture and destabilization.

Because of these processes, overall vascular ho- (45), endothelial cell inflammation (39) , and smooth muscle cell migration and proliferation (46, 47) . Accordingly, genetic deletion of Tlr2 in atherosclerotic mice reduced the severity of disease and aggregation of foam cells in the aorta (48) . Tlr1 À/À and Tlr6 À/À mice with atherosclerosis exhibited the same degree of disease severity as control atherosclerotic mice (49) , which indicated that TLR2 might play a role in atherosclerosis as a heterodimer in contrast to TLR1 and TLR6, which do not seem to be atherogenic per se. In this context, it would be interesting to evaluate the consequences of the inhibition of both TLR1 and TLR6 to test whether a compensatory mechanism exists between the heterodimers. Endosomal TLRs have also been associated with atherosclerosis, but published studies are contradictory or inconclusive as to whether they play protective or deleterious roles. For instance, TLR3 expression seems to be upregulated in atheromas, and its activation increases plaque size (50) . However, Tlr3 À/À Apoe À/À mice develop atherosclerosis earlier than Tlr3 þ/þ Apoe À/À mice (24) , and further stimulation of TLR3 in this model reduces the formation of scar tissue (51) , which suggests a protective effect for this TLR. Likewise, TLR7 and TLR9 appear to play protective roles because lipid aggregation, macrophage infiltration and accumulation, and production of pro-inflammatory cytokines are exacerbated in both Tlr7 À/À Apoe À/À and Tlr9 À/À Apoe À/À mice (52) (53) (54) (60) . Several experimental models broadly described the contribution of TLR4 to hypertension progression (61, 62) . In addition to TLR4, TLR9 activation was found to increase blood pressure, leading to vascular alterations in normotensive rats (63) and

triggering hypertension in pregnant rats (64) . TLR9 inhibition was also found to reduce blood pressure in hypertensive rats (65) .

TLR4 upregulation was also widely associated with abdominal aortic aneurysm; its inhibition prevented aortic dilation and aneurysm progression in several experimental models (59, 66, 67) . Inhibition of TLR2 also ameliorated abdominal aortic aneurysm in mice (68) , and its genetic deletion was found to prevent abdominal aortic aneurysm aggravated by periodontal bacterial infection (69) . TLR3 also seems to be upregulated in aortic wall tissue during abdominal aortic aneurysm development; however, its specific involvement in this pathology remains unclear (70) . Restoration of blood flow to ischemic myocardium is associated with cardiac injury mainly due to the toxic effects of reactive oxygen intermediates that are generated during heart reperfusion (72 inflammasome (113, 114) . NEK7 deficiency reduces IL-1b production and disease severity in murine models (106, 114) . (132) . This increase has been observed both in foam cells and in endothelial cells (132) . In addition, NLRP3 appears to be upregulated in the aorta of patients with atherosclerosis (133) . However, studies performed in atherosclerosis-prone mice yielded conflicting results as to the participation of the NLRP3 inflammasome.

Although some studies reported that the deletion of Nlrp3, Asc, Casp1, or Il1a/b attenuated lesion development (116, 134) , diminished macrophage infiltration (135) , and decreased the severity of the disease (136),

another study failed to observe any significant change in atherosclerosis development or macrophage infiltration upon deletion of any of these genes (137) . These findings seem to be highly dependent on the animal model chosen (Ldlr À/À or Apoe À/À ) and Based on the results of these pre-clinical studies, 2 clinical trials were designed to investigate targeting the NLRP3 inflammatory cascade in patients with AMI:

1) a trial on anakinra, which is a recombinant IL-1b receptor antagonist; and 2) a trial on canakinumab, which is a monoclonal antibody targeting IL-1b. Both trials are in phase II and phase III, respectively (116, 154) . Both trials reported a decrease in cardiovascular risk with treatment; however, further research is needed to determine effective doses and to ascertain and reduce the associated side effects (116, 154) .

HEART FAILURE. Research on the involvement of the NLRP3 inflammasome in HF is more scarce, with only a few studies describing an increase in both IL-1b (155, 156) and IL-18 (157) (165) (166) (167) . Since then, the NOD family has widened; currently, it consists of >20 human proteins, together with a large number of proteins from animals, plants, bacteria, and fungi (167) .

As alluded to earlier, Nod1 encodes an intracellular scaffolding protein that consists of CARD, NOD, and leucine-rich repeat domains ( Figure 1C) . pathway, which suggests a potential role for NOD1 in inflammatory diseases associated with this perturbation. The link between NOD1 and ER stress appears to be the unfolded protein response, as inhibiting IRE1a, a kinase implicated in this pathway, attenuates the NOD1-associated inflammatory reaction (170) . Some studies have associated ER stress with an imbalance in cellular Ca 2þ and the activation of NOD1 signaling (171, 172) . A recent study revealed that NOD1 activation both by bacterial pathogens and the NOD ligand to the scientific community, blossomed into a profitable business model that, like all businesses, had to compete for articles and readers in order to stay in business.

As journals evolved and expanded, journal editors recognized that to continue to flourish and maintain market share, they needed to have a broader appeal to their readership that extended beyond publishing scientific articles, which led to journals to publish timely reviews and letters that informed the readership on the latest developments in their respective fields. Indeed, Sir Theodore ("Robbie") Fox, the prolific and transformative editor of the Lancet (1944 to 1964) would often refer to the Lancet as a newspaper (4) . Pari passu with the transition of journals from being a gateway for reporting the latest scientific articles to providing timely newsworthy content to their readers, in the 1990s, journals also began to simultaneously publish articles on late-breaking clinical trials that were being presented at national meetings. Whereas the New England Journal of Medicine has been always been particularly adept at attracting topline clinical trials presented at national meetings, it is important to note that all of the major journals adopted this practice to remain competitive within this specific journalistic space. As noted by Larry Husten, a veteran journalist who has covered cardiology news for decades, "simultaneous publication.brings a lot of benefits, including more rigorous peer review and superior and more widespread availability of trial results. Reducing the time between initial presentation and publication helps shorten the gap between the acquisition of new knowledge and its application to clinical practice."

However, Husten also notes that simultaneous publication of clinical trials has "unintended consequences," including "the publication barrier seems to be far lower when a trial is published simultaneously at a major medical meeting." He also states that clinical trialists have confided to him that "compared to non-meeting related publications, the peer review process will be both less rigorous and less prolonged" (6) . Although hard data are lacking with respect to whether the peer review process is actually less rigorous for articles that are submitted for simultaneous publications, the editorial process surrounding late-breaking clinical trials changed and became more streamlined, given the competing pressures imposed by the abbreviated time frames for peer review, for statistical review, for editorial oversight, and for generation of fully formatted articles that were available online at the time of the presentation at a meeting. Whereas journals have been remarkably successful at accomplishing these tasks year after

year, the need for speed in publishing changed the way in which the editorial process works, which laid the groundwork, at least in part, for the way for the way in which journals have faced the challenges imposed by COVD-19.

As the sheer magnitude of the COVID-19 pandemic began to become apparent, the pace of scientific publishing accelerated rapidly, dramatically com- papers had been retracted, 2 had been temporarily retracted, and 2 papers have been cited as containing misleading information (i.e., expressions of concern) (8). By the time this Editor's Page appears in its final form, the number of COVID-19 retractions will almost assuredly have increased.

In a remarkably prescient article titled "The 

Heart disease and stroke statistics-2020 update: a report from the

Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials

Is cardioprotection dead?

New and revisited approaches to preserving the reperfused myocardium

Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy

Dietary coenzyme Q(10) supplement renders swine hearts resistant to ischemia-reperfusion injury

Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion

Effect of cyclosporine on reperfusion injury in acute myocardial infarction

Cyclosporine before PCI in patients with acute myocardial infarction

A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Abeta

Humanin peptide suppresses apoptosis by interfering with Bax activation

Humanin inhibits neuronal cell death by interacting with a cytokine receptor complex or complexes involving CNTF receptor alpha/WSX-1/ gp130

Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis

The emerging role of the mitochondrial-derived peptide humanin in stress resistance

Potent humanin analog increases glucose-stimulated insulin secretion through enhanced metabolism in the beta cell

The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus

from cardiovascular magnetic resonance

Achieving door-to-balloon times that meet quality guidelines: how do successful hospitals do it?

Cardioprotection: where to from here?

Ischemic preconditioning at a distance

Rapid surface cooling by ThermoSuit system dramatically reduces scar size, prevents post-infarction adverse left ventricular remodeling, and improves cardiac function in rats

Myocardial hypothermia

Enhancement of salvage of reperfused myocardium by early beta-adrenergic blockade (timolol)

The wavefront phenomenon of ischemic cell death. 1. Myocardial infarct size vs duration of coronary occlusion in dogs

Role of apoptosis in reperfusion injury

Signalling pathways and mechanisms of protection in pre-and

Assay of endopeptidase-24.11 activity in plasma applied to in vivo studies of endopeptidase inhibitors

Elevated plasma B-type natriuretic peptide concentrations directly inhibit circulating neprilysin activity in heart failure

Do plasma neprilysin activity and plasma neprilysin concentration predict cardiac events in chronic kidney disease patients?

Neprilysin levels in plasma and synovial fluid of juvenile idiopathic arthritis patients

Neprilysin, obesity and the metabolic syndrome

Roles of neutral endopeptidase in airways

Ab-degrading enzymes: potential for treatment of Alzheimer disease

Improved glycemia in high-fat-fed neprilysin-deficient mice is associated with reduced DPP-4 activity and increased active GLP-1 levels

Soluble neprilysin is predictive of cardiovascular death and heart failure hospitalization in heart failure patients

Prognostic value and kinetics of soluble neprilysin in acute heart failure: a pilot study

Soluble neprilysin retains catalytic activity in heart failure

Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research

Sacubitril/valsartan: beyond natriuretic peptides

Prognostic implications of changes in N-terminal pro-B-type natriuretic peptide in patients with heart failure

Long-term neprilysin inhibitionimplications for ARNIs

Neutral endopeptidase 24.11 in rat peripheral tissues: comparative localization by 'ex vivo' and 'in vitro' autoradiography

Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics

The heart regulates the endocrine response to heart failure: cardiac contribution to circulating neprilysin

Effects of hypoxia and oxidative stress on expression of neprilysin in human neuroblastoma cells and rat cortical neurones and astrocytes

Androgens regulate neprilysin expression: role in reducing b-amyloid levels

Estrogen stimulates degradation of b-amyloid peptide by upregulating neprilysin

Somatostatin regulates brain amyloid b peptide Ab42 through modulation of proteolytic degradation

Impact of vitamin D on amyloid precursor protein processing and amyloid-b peptide degradation in Alzheimer's disease

Increased expression of renal neutral endopeptidase in severe heart failure

Effects of sacubitril/valsartan on neprilysin targets and the metabolism of natriuretic peptides in chronic heart failure: a mechanistic clinical study

Heart failure with reduced ejection fraction is characterized by systemic NEP down-regulation

Pulmonary and renal neutral endopeptidase EC 3.4.24.11 in rats with experimental heart failure

Discrepancy between plasma and lung angiotensin-converting enzyme activity in experimental congestive heart failure. A novel aspect of endothelium dysfunction

Renal response to acute neutral endopeptidase inhibition in mild and severe experimental heart failure

Differential regulation of ANP and BNP in acute decompensated heart failure: deficiency of ANP

What do you have to lose?' How Trump has promoted malaria drug. The New York Times

A mysterious company's coronavirus papers in top medical journals may be unraveling

Retraction-hydroxychloroquine or chloroquine with or without macrolide for treatment of COVID-19: a multinational registry analysis

Retraction-cardiovascular disease, drug therapy, and mortality in Covid-19

Researcher involved in retracted Lancet study has faculty appointment terminated, as details in scandal emerge

A randomized trial of hydroxychloroquine as postexposure prophylaxis for Covid-19

Trump administration announces framework and leadership for 'Operation Warp Speed

Plotkin's Vaccines. 7th edition

Trump administration's Operation Warp Speed accelerates AstraZeneca COVID-19 vaccine to be available beginning in October

Developing Covid-19 vaccines at pandemic speed

How long will a vaccine really take? The New York Times

mcm-legal-regulatory-and-policyframework/emergency-use-authorization

Plotkin's Vaccines. 7th Edition

Val-Blasco, and Prieto contributed equally to this work and are joint first authors. This work was supported by the Spanish Ministry of Economy and Competitiveness (AEI/EU) and FEDER: SAF2017-82436R and RTC2017-6283)

Jaén holds a FPU fellowship from MECD (Spain). Dr. López-Sendón has received research grants from Sanofi

Tolerance, danger, and the extended family

NF-kappaB and the immune response

Pattern recognition receptors and inflammation

Newly described pattern recognition receptors team up against intracellular pathogens

Innate recognition of microbial-derived signals in immunity and inflammation

Self-regulation and cross-regulation of pattern-recognition receptor signalling in health and disease

Sterile inflammation: sensing and reacting to damage

DAMP-sensing receptors in sterile inflammation and inflammatory diseases

Emerging insights into molecular mechanisms underlying pyroptosis and functions of inflammasomes in diseases

Insights into innate immune signalling in controlling cardiac remodelling

Innate immunity and the failing heart

The infarcted myocardium solicits GM-CSF for the detrimental oversupply of inflammatory leukocytes

Targeting interleukin-1b reduces leukocyte production after acute myocardial infarction

Myocardial infarction activates CCR2(þ) hematopoietic stem and progenitor cells

Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1

The role of toll-like receptor signaling in the progression of heart failure

Toll-like receptors: significance, ligands, signaling pathways, and functions in mammals

Toll-like receptor signaling pathways

Nucleic acidsensing TLRs: trafficking and regulation

Toll-like receptors in innate immunity

Toll-like receptors

Tolllike receptors in atherosclerosis

The role of patternrecognition receptors in innate immunity: update on toll-like receptors

MyD88: a central player in innate immune signaling

TLR-signaling networks: an integration of adaptor molecules, kinases, and cross-talk

TLRs and chronic inflammation

The role of toll-like receptors in chronic inflammation

Role of pattern-recognition receptors in cardiovascular health and disease

Role of toll-like receptors in cardiovascular diseases

TLR signalling and association of TLR polymorphism with cardiovascular diseases

Anti-inflammatory therapies for atherosclerosis

Omega-3 fatty acids in atherosclerosis and coronary artery disease

Inflammation in atherosclerosis: transition from theory to practice

A gift from nature: the birth of the statins

Toll-like receptors and atherosclerosis: oxidized LDL as an endogenous toll-like receptor ligand

Endothelialvascular smooth muscle cells interactions in atherosclerosis

Expression of toll-like receptors in human atherosclerotic lesions: a possible pathway for plaque activation

atherosclerotic plaques: a novel effect of statins

Overexpression of toll-like receptors 2, 3, 4, and 8 is correlated to the vascular atherosclerotic process in the hyperlipidemic rabbit model: the effect of statin treatment

Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development

Adipocyte fatty acid-binding protein

Atherosclerosis induced by endogenous and exogenous toll-like receptor (TLR)1 or TLR6 agonists

Activation of endothelial toll-like receptor 3 impairs endothelial function

Unexpected protective role for toll-like receptor 3 in the arterial wall

Pathogen-sensing plasmacytoid dendritic cells stimulate cytotoxic T-cell function in the atherosclerotic plaque through interferon-alpha

Toll-like receptor 7 protects from atherosclerosis by constraining 'inflammatory' macrophage activation

Protective role for toll-like receptor-9 in the development of atherosclerosis in apolipoprotein Edeficient mice

Blocking toll-like receptors 7 and 9 reduces postinterventional remodeling via reduced macrophage activation, foam cell formation, and migration

Toll-like receptor 9 plays a pivotal role in angiotensin IIinduced atherosclerosis

Toll-like receptor 9 inactivation alleviated atherosclerotic progression and inhibited macrophage polarized to M1 phenotype in ApoE-/-mice

Tolllike receptors in the vascular system: sensing the dangers within

Alginate oligosaccharide indirectly affects toll-like receptor signaling via the inhibition of microRNA-29b in aneurysm patients after endovascular aortic repair

The interplay between angiotensin II, TLR4 and hypertension

Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension

Tolllike receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats

Circulating mitochondrial DNA and toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats

Toll-like receptor 9 activation: a novel mechanism linking placenta-derived mitochondrial DNA and vascular dysfunction in pre-eclampsia

The role of toll-like receptors and vitamin D in cardiovascular diseases-a review

Tanshinone IIA attenuates elastase-induced AAA in rats via inhibition of MyD88-dependent TLR-4 signaling

Rosiglitazone negatively regulates c-Jun N-terminal kinase and toll-like receptor 4 proinflammatory signalling during initiation of experimental aortic aneurysms

Antagonism of toll-like receptor 2 attenuates the formation and progression of abdominal aortic aneurysm

Toll-like receptor-2 plays a fundamental role in periodontal bacteria-accelerated abdominal aortic aneurysms

Analysis of host toll-like receptor 3 and RIG-I-like receptor gene expression in patients with abdominal aortic aneurysm

Epidemiology of myocardial infarction

Myocardial ischemia-reperfusion injury: a neglected therapeutic target

Unstable angina activates myocardial heat shock protein 72, endothelial nitric oxide synthase, and transcription factors NFkappaB and AP-1

Hsp 70, hsCRP and oxidative stress in patients with acute coronary syndromes

TLR4 mutation and HSP60-induced cell death in adult mouse cardiac myocytes

Extracellular heat shock protein 60, cardiac myocytes, and apoptosis

Mechanism of TLR-4/NF-kB pathway in myocardial ischemia reperfusion injury of mouse

Myocardial ischemia/reperfusion injury is mediated by leukocytic toll-like receptor-2 and reduced by systemic administration of a novel anti-toll-like receptor-2 antibody

Inhibition of toll-like receptor 4 with eritoran attenuates myocardial ischemia-reperfusion injury

Early atorvastatin therapy improves cardiac function in patients with acute myocardial infarction

Tolllike receptor-2 modulates ventricular remodeling after myocardial infarction

Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function after myocardial infarction

Role of extracellular RNA and TLR3-Trif signaling in myocardial ischemia-reperfusion injury

Innate immunity mediates myocardial preconditioning through toll-like receptor 2 and TIRAP-dependent signaling pathways

CpG-ODN, the TLR9 agonist, attenuates myocardial ischemia/reperfusion injury: involving activation of PI3K/Akt signaling

Global public health burden of heart failure

Inhibition of tolllike receptor signaling as a promising therapy for inflammatory diseases: a journey from molecular to nano therapeutics

Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium

Increased toll-like receptor 4 in the myocardium of patients requiring left ventricular assist devices

The toll-like receptor 4-antagonist eritoran reduces murine cardiac hypertrophy

Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure

Cardiac excitation-contraction coupling

Extracellular heat shock protein 70 induces cardiomyocyte inflammation and contractile dysfunction via TLR2

Hemorrhagic shock induces NAD(P)H oxidase activation in neutrophils: role of HMGB1-TLR4 signaling

High-mobility

Toll-like receptor 4 knockout protects against anthrax lethal toxin-induced cardiac contractile dysfunction: role of autophagy

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2þ handling via a NADPH oxidase-dependent mechanism

Sensing and reacting to microbes through the inflammasomes

Intracellular NOD-like receptors in host defense and disease

NODs: intracellular proteins involved in inflammation and apoptosis

Nucleotidebinding oligomerization domain-like receptors and inflammasomes in the pathogenesis of nonmicrobial inflammation and diseases

Mechanism and regulation of NLRP3 inflammasome activation

Targeting the NLRP3 inflammasome in inflammatory diseases

Gasdermin: a new player to the inflammasome game

Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis

Non-transcriptional priming and deubiquitination regulate NLRP3 inflammasome activation

K þ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter

NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component

NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals

Antiinflammatory therapy with canakinumab for atherosclerotic disease

Targeting interleukin-1 in heart disease

Interleukin 18 in the heart

IL-1beta and TNF-alpha upregulate angiotensin II type 1 (AT1) receptors on cardiac fibroblasts and are associated with increased AT1 density in the post-MI heart

IL-18 cleavage triggers cardiac inflammation and fibrosis upon badrenergic insult

IL-1b and IL-18: inflammatory markers or mediators of hypertension

Targeting interleukin-1 in heart failure and inflammatory heart disease

Circulating levels of interleukin 18 reflect etiologies of heart failure: Th1/Th2 cytokine imbalance exaggerates the pathophysiology of advanced heart failure

Enhanced cardiomyocyte NLRP3 inflammasome signaling promotes atrial fibrillation

NLRP3 and downstream cytokine expression elevated in the monocytes of patients with coronary artery disease

Upregulated NLRP3 inflammasome activation in patients with type 2 diabetes

Role of NLRP3 inflammasomes in atherosclerosis

Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling

Paramel Varghese G, Folkersen L, Strawbridge RJ, et al. NLRP3 inflammasome expression and activation in human atherosclerosis

Correlation of NLRP3 with severity and prognosis of coronary atherosclerosis in acute coronary syndrome patients

Novel insights into the NLRP 3 inflammasome in atherosclerosis

Caspase-1 deficiency decreases atherosclerosis in apolipoprotein E-null mice

Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western dietfed apolipoprotein E-deficient mice

Lack of interleukin-1beta decreases the severity of atherosclerosis in ApoE-deficient mice

Statins attenuate activation of the NLRP3 inflammasome by oxidized LDL or TNFa in vascular endothelial cells through a PXR-dependent mechanism

NLRP3 inflammasome activation in coronary artery disease: results from prospective and randomized study of treatment with atorvastatin or rosuvastatin

Is immunity a mechanism contributing to statin-induced diabetes?

Fluvastatin causes NLRP3 inflammasome-mediated adipose insulin resistance

Inhibition of interleukin-1b decreases aneurysm formation and progression in a novel model of thoracic aortic aneurysms

Inflammasome activity in leucocytes decreases with abdominal aortic aneurysm progression

The selective NLRP3-inflammasome inhibitor

Inflammasome activation of cardiac fibroblasts is essential for myocardial ischemia/reperfusion injury

The NLRP3 inflammasome in acute myocardial infarction

Gastrodin ameliorates microvascular reperfusion injury-induced pyroptosis by regulating the NLRP3/caspase-1 pathway

Protective effect of ethyl pyruvate against myocardial ischemia reperfusion injury through regulations of ROS-related NLRP3 inflammasome activation

Naoxintong attenuates ischaemia/reperfusion injury through inhibiting NLRP3 inflammasome activation

Rationale and design of the Virginia Commonwealth University-Anakinra Remodeling Trial-3 (VCU-ART3): a randomized, placebocontrolled, double-blinded, multicenter study

A Review of interleukin-1 in heart disease: where do we stand today?

Enhanced interleukin-1 activity contributes to exercise intolerance in patients with systolic heart failure

Evidence for altered interleukin 18 (IL)-18 pathway in human heart failure

Formation of the inflammasome in acute myocarditis

The importance of NLRP3 inflammasome in heart failure

Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome

Cloning and characterization of an extracellular Ca2þ-sensing receptor from bovine parathyroid

The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2þ and cAMP

Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure

Cardiomyocyte inflammasome signaling in cardiomyopathies and atrial fibrillation: mechanisms and potential therapeutic implications

The molecular mechanism of programmed cell death in C. elegans

Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance

The NOD: a signaling module that regulates apoptosis and host defense against pathogens

Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kB

NOD1 and NOD2: signaling, host defense, and inflammatory disease

NOD1 and NOD2 signalling links ER stress with inflammation

Activation of the endoplasmic reticulum stress response impacts the NOD1 signaling pathway

Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages

Coordinated augmentation of NFAT and NOD signaling mediates proliferative VSMC phenotype switch under hyperinsulinemia

Activation of an innate immune receptor, Nod1, accelerates atherogenesis in Apoe À/À mice

NOD1 receptor is up-regulated in diabetic human and murine myocardium

Activation of NOD1 by DAP contributes to myocardial ischemia/ reperfusion injury via multiple signaling pathways

NOD1, a new player in cardiac function and calcium handling

Deficiency of NOD1 improves the b-adrenergic modulation of Ca2þ handling in a mouse model of heart failure

KEY WORDS cardiovascular disease, innate immune system, NLRP3, NOD1, nucleotidebinding oligomerization domain-like receptors, toll-like receptors

Situ immune profiling of heart transplant biopsies improves diagnostic accuracy and rejection risk stratification

Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection

Allograft rejection of ISHLT grade $3A occurring late after heart transplantation-a distinct entity?

Effect on long-term mortality of HLA-DR matching in heart transplantation

Fluid-based assays and precision medicine of cardiovascular diseases: the "hope" for Pandora's box?

Situ immune profiling of heart transplant biopsies improves diagnostic accuracy and rejection risk stratification

Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection

Allograft rejection of ISHLT grade $3A occurring late after heart transplantation-a distinct entity?

Effect on long-term mortality of HLA-DR matching in heart transplantation

Fluid-based assays and precision medicine of cardiovascular diseases: the "hope" for Pandora's box?

RETRACTED: Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis

Retraction: Cardiovascular disease, drug therapy, and mortality in Covid-19

The trouble with medical journals

Table of Contents of Philosophical Transactions of the Royal Society of London. 1665. Available at

Hot Lines, Simultaneous Publications, and the Decline of Medical Meetings

World Health Organization. Novel Coronavirus (2019-nCoV): Situation Report-13

Retracted Coronavirus (COVID-19) Papers. 2020

The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit

Please note: The authors have reported that they have no relationships relevant to the contents of this paper to disclose.The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the JACC: Basic to Translational Science author instructions page.Please note: The authors have reported that they have no relationships relevant to the contents of this paper to disclose.The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the JACC: Basic to Translational Science author instructions page.