DNA-sensing inflammasomes cause recurrent atherosclerotic stroke.

The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies1. Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events1,2. However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events.

RAAS inhibition and beyond-cardiovascular medications in patients at risk of or affected by COVID-19.

The COVID-19 pandemic led to an enormous burden on healthcare systems worldwide. Causal therapy is still in its infancy. Contrary to initial views that the use of angiotensin-converting enzyme inhibitors (ACEi)/angiotensin II receptor blockers (ARBs) may increase the risk for a deleterious disease course, it has been shown that these agents may actually be beneficial for patients affected by COVID-19. In this article, we provide an overview of the three most commonly used classes of drugs in cardiovascular disease (ACEi/ARB, statins, beta-blockers) and their potential role in COVID-19 therapy. More results from randomized clinical trials are necessary to identify patients that can benefit most from the use of the respective drugs.

The Impact of Exercise on Immunity, Metabolism, and Atherosclerosis.

Physical exercise represents an effective preventive and therapeutic strategy beneficially modifying the course of multiple diseases. The protective mechanisms of exercise are manifold; primarily, they are elicited by alterations in metabolic and inflammatory pathways. Exercise intensity and duration strongly influence the provoked response. This narrative review aims to provide comprehensive up-to-date insights into the beneficial effects of physical exercise by illustrating the impact of moderate and vigorous exercise on innate and adaptive immunity. Specifically, we describe qualitative and quantitative changes in different leukocyte subsets while distinguishing between acute and chronic exercise effects. Further, we elaborate on how exercise modifies the progression of atherosclerosis, the leading cause of death worldwide, representing a prime example of a disease triggered by metabolic and inflammatory pathways. Here, we describe how exercise counteracts causal contributors and thereby improves outcomes. In addition, we identify gaps that still need to be addressed in the future.

Comparison of the effects of a truncating and a missense MYBPC3 mutation on contractile parameters of engineered heart tissue.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. The most frequently mutated gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373_2374insG) and a missense mutation (c.1591G>C) in MYBPC3 in engineered heart tissue (EHT). EHTs enable to study the direct effects of mutants without interference of secondary disease-related changes. EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO EHTs displayed higher maximal force and sensitivity to external [Ca(2+)] than WT EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. In conclusion, this study provides evidence in KO EHTs that i) haploinsufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤73%, ii) missense or truncating mutations, but not WT do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated.

Multi-Omic Candidate Screening for Markers of Severe Clinical Courses of COVID-19.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) disease courses are characterized by immuno-inflammatory, thrombotic, and parenchymal alterations. Prediction of individual COVID-19 disease courses to guide targeted prevention remains challenging. We hypothesized that a distinct serologic signature precedes surges of IL-6/D-dimers in severely affected COVID-19 patients. METHODS: We performed longitudinal plasma profiling, including proteome, metabolome, and routine biochemistry, on seven seropositive, well-phenotyped patients with severe COVID-19 referred to the Intensive Care Unit at the German Heart Center. Patient characteristics were: 65 ± 8 years, 29% female, median CRP 285 ± 127 mg/dL, IL-6 367 ± 231 ng/L, D-dimers 7 ± 10 mg/L, and NT-proBNP 2616 ± 3465 ng/L. RESULTS: Based on time-series analyses of patient sera, a prediction model employing feature selection and dimensionality reduction through least absolute shrinkage and selection operator (LASSO) revealed a number of candidate proteins preceding hyperinflammatory immune response (denoted ΔIL-6) and COVID-19 coagulopathy (denoted ΔD-dimers) by 24-48 h. These candidates are involved in biological pathways such as oxidative stress/inflammation (e.g., IL-1alpha, IL-13, MMP9, C-C motif chemokine 23), coagulation/thrombosis/immunoadhesion (e.g., P- and E-selectin), tissue repair (e.g., hepatocyte growth factor), and growth factor response/regulatory pathways (e.g., tyrosine-protein kinase receptor UFO and low-density lipoprotein receptor (LDLR)). The latter are host- or co-receptors that promote SARS-CoV-2 entry into cells in the absence of ACE2. CONCLUSIONS: Our novel prediction model identified biological and regulatory candidate networks preceding hyperinflammation and coagulopathy, with the most promising group being the proteins that explain changes in D-dimers. These biomarkers need validation. If causal, our work may help predict disease courses and guide personalized treatment for COVID-19.

Association of In-Hospital Hemoglobin Drop With Decreased Myocardial Salvage and Increased Long-Term Mortality in Patients With Acute ST-Segment-Elevation Myocardial Infarction.

Background Anemia and blood loss occur often in patients with ST-segment-elevation myocardial infarction (STEMI). In-hospital hemoglobin drop is associated with 1-year mortality in patients with acute coronary syndrome. However, data on the effect of hemoglobin reduction on myocardial salvage and long-term outcomes are scarce. We investigated the impact of in-hospital hemoglobin drop on myocardial salvage and 5-year mortality in patients with STEMI treated with primary percutaneous coronary intervention. Methods and Results In-hospital hemoglobin drop was defined as a decrease in hemoglobin levels from admission and nadir hemoglobin values. Patients were categorized as having the following: no drop, minimal drop (<3 g/dL), minor drop (≥3 to <5 g/dL), and major drop (≥5 g/dL). Myocardial area at risk and infarct size were measured using serial single-photon emission computerized tomography imaging. The co-primary outcomes were myocardial salvage and 5-year all-cause mortality. Of 1204 patients, 1169 (97.1%) showed a hemoglobin drop during hospitalization: minimal, minor, and major drop occurred in 894 (74.3%), 214 (17.8%), and 61 (5.1%) patients, respectively. Myocardial salvage was reduced in patients with minimal (median, 0.53 [interquartile range, 0.27-0.83]), minor (median, 0.40 [interquartile range, 0.18-0.62]), and major (median, 0.40 [interquartile range, 0.14-0.77]) drop compared with patients without drop (median, 0.70 [interquartile range, 0.44-1.0], P<0.001). After adjusting for covariates, hemoglobin drop remained an independent correlate of poor myocardial salvage. A drop of ≥3 g/dL was associated with reduced left ventricular function at 6 months and with increased mortality at 5-year follow-up after STEMI. Conclusions In patients with STEMI undergoing primary percutaneous coronary intervention, in-hospital hemoglobin drop was associated with reduced myocardial salvage, left ventricular function, and increased long-term mortality.

Colchicine Impacts Leukocyte Trafficking in Atherosclerosis and Reduces Vascular Inflammation.

Background: Inflammation strongly contributes to atherosclerosis initiation and progression. Consequently, recent clinical trials pharmacologically targeted vascular inflammation to decrease the incidence of atherosclerosis-related complications. Colchicine, a microtubule inhibitor with anti-inflammatory properties, reduced cardiovascular events in patients with recent acute coronary syndrome and chronic coronary disease. However, the biological basis of these observations remains elusive. We sought to explore the mechanism by which colchicine beneficially alters the course of atherosclerosis. Methods and Results: In mice with early atherosclerosis (Apoe-/- mice on a high cholesterol diet for 8 weeks), we found that colchicine treatment (0.25 mg/kg bodyweight once daily over four weeks) reduced numbers of neutrophils, inflammatory monocytes and macrophages inside atherosclerotic aortas using flow cytometry and immunohistochemistry. Consequently, colchicine treatment resulted in a less inflammatory plaque composition and reduced plaque size. We next investigated how colchicine prevented plaque leukocyte expansion and found that colchicine treatment mitigated recruitment of blood neutrophils and inflammatory monocytes to plaques as revealed by adoptive transfer experiments. Causally, we found that colchicine reduced levels of both leukocyte adhesion molecules and receptors for leukocyte chemoattractants on blood neutrophils and monocytes. Further experiments showed that colchicine treatment reduced vascular inflammation also in post-myocardial infarction accelerated atherosclerosis through similar mechanisms as documented in early atherosclerosis. When we examined whether colchicine also decreased numbers of macrophages inside atherosclerotic plaques by impacting monocyte/macrophage transitioning or in-situ proliferation of macrophages, we report that colchicine treatment did not influence macrophage precursor differentiation or macrophage proliferation using cell culture experiments with bone marrow derived macrophages. Conclusions: Our data reveal that colchicine prevents expansion of plaque inflammatory leukocytes through lowering recruitment of blood myeloid cells to plaques. These data provide novel mechanistic clues on the beneficial effects of colchicine in the treatment of atherosclerosis and may inform future anti-inflammatory interventions in patients at risk.

Circulating Nucleosomes as Potential Markers to Monitor COVID-19 Disease Progression.

The severity of coronavirus disease 2019 (COVID-19) varies significantly with cases spanning from asymptomatic to lethal with a subset of individuals developing Severe Acute Respiratory Syndrome (SARS) and death from respiratory failure. To determine whether global nucleosome and citrullinated nucleosome levels were elevated in COVID-19 patients, we tested two independent cohorts of COVID-19 positive patients with quantitative nucleosome immunoassays and found that nucleosomes were highly elevated in plasma of COVID-19 patients with a severe course of the disease relative to healthy controls and that both histone 3.1 variant and citrullinated nucleosomes increase with disease severity. Elevated citrullination of circulating nucleosomes is indicative of neutrophil extracellular trap formation, neutrophil activation and NETosis in severely affected individuals. Importantly, using hospital setting (outpatient, inpatient or ICU) as a proxy for disease severity, nucleosome levels increased with disease severity and may serve as a guiding biomarker for treatment. Owing to the limited availability of mechanical ventilators and extracorporal membrane oxygenation (ECMO) equipment, there is an urgent need for effective tools to rapidly assess disease severity and guide treatment selection. Based on our studies of two independent cohorts of COVID-19 patients from Belgium and Germany, we suggest further investigation of circulating nucleosomes and citrullination as biomarkers for clinical triage, treatment allocation and clinical drug discovery.

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue.

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca2+] under pacing. Adeno-associated virus-mediated gene transfer of D282 and S282 similarly restored Mybpc3 mRNA and protein levels and suppressed mutant Mybpc3 transcripts. Moreover, both exogenous cMyBP-C proteins were properly incorporated in the sarcomere. KI EHTs hypercontractility was similarly prevented by both treatments, but S282 had a stronger effect than D282 to normalize the force-Ca2+-relationship and the expression of dysregulated genes. These findings in an in vitro model indicate that S282 is a better choice than D282 to restore the HCM EHT phenotype. To which extent the results apply to human HCM remains to be seen.

Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice.

Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.