Serum amyloid A-A potential therapeutic target for hyper-inflammatory syndrome associated with COVID-19.

Serum amyloid-A (SAA) is associated with inflammatory disorders such as rheumatoid arthritis, Familial Mediterranean Fever, sarcoidosis, and vasculitis. There is accumulating evidence that SAA is a reliable biomarker for these autoinflammatory and rheumatic diseases and may contribute to their pathophysiology. Hyperinflammatory syndrome associated with COVID-19 is a complex interaction between infection and autoimmunity and elevation of SAA is strongly correlated with severity of the inflammation. In this review we highlight the involvement of SAA in these different inflammatory conditions, consider its potential role and discuss whether it could be a potential target for treatment of the hyperinflammatory state of COVID-19 with many potential advantages and fewer adverse effects. Additional studies linking SAA to the pathophysiology of COVID-19 hyper-inflammation and autoimmunity are needed to establish the causal relationship and the therapeutic potential of inhibitors of SAA activity.

Increased Expression Levels of Netrin-1 in Visceral Adipose Tissue during Obesity Favour Colon Cancer Cell Migration.

Netrin (NTN)-1, an extracellular matrix protein with a crucial role in inflammation, is dysregulated during obesity (OB) and influences colon cancer (CC) progression. To decipher the mechanisms underlying CC development during obesity, we examined the expression of NTN1 and its receptors in the visceral adipose tissue (VAT) of 74 (25 normal weight (NW)) (16 with CC) and 49 patients with OB (12 with CC). We also evaluated the effect of caloric restriction (CR) on the gene expression levels of Ntn1 and its receptors in the colon from a rat model fed a normal diet. The impact of adipocyte-conditioned media (ACM) from patients with OB and NTN-1 was assessed on the expression levels of neogenin 1(NEO1), deleted in colorectal carcinomas (DCC) and uncoordinated-5 homolog B (UNC5B) in Caco-2 and HT-29 human colorectal cell lines, as well as on Caco-2 cell migration. Increased NTN1 and NEO1 mRNA levels in VAT were due to OB (p < 0.05) and CC (p < 0.001). In addition, an upregulation in the expression levels of DCC and UNC5B in patients with CC (p < 0.01 and p < 0.05, respectively) was observed. Decreased (p < 0.01) Ntn1 levels in the colon from rats submitted to CR were found. In vitro experiments showed that ACM increased DCC (p < 0.05) and NEO1 (p < 0.01) mRNA levels in HT-29 and Caco-2 cell lines, respectively, while UNC5B decreased (p < 0.01) in HT-29. The treatment with NTN-1 increased (p < 0.05) NEO1 mRNA levels in HT-29 cells and DCC (p < 0.05) in both cell lines. Finally, we revealed a potent migratory effect of ACM and NTN-1 on Caco-2 cells. Collectively, these findings point to increased NTN-1 during OB and CC fuelling cancer progression and exerting a strong migratory effect on colon cancer cells.

Role of platelet function testing in acute coronary syndromes: a meta-analysis.

OBJECTIVE: This meta-analysis aimed to evaluate whether using platelet function testing (PFT) in acute coronary syndromes (ACS) to personalise antiplatelet therapy including a P2Y12 antagonist offers any clinical benefits to indicate incorporation into routine practice. METHODS: A search was conducted on five databases for randomised controlled trials (RCTs) conducted between 1 January 2000 and 17 July 2022, which included an ADP-specific platelet function assays and P2Y12 antagonists as part of dual antiplatelet therapy (DAPT) and have reported the efficacy and/or safety outcomes. The reported event frequencies were used to calculate the risk ratios (RRs) with a 95% CI. The χ2 heterogeneity statistical test and sensitivity analysis were used for heterogeneity assessment. RESULTS: Five RCTs with 7691 patients were included in the analysis. No significant risk reduction was seen in major adverse cardiovascular events (RR=0.95, p=0.42), individual cardiac events (cardiovascular death: RR=0.76, p=0.26; myocardial infarction: RR=0.96, p=0.74; stent thrombosis: RR=0.92, p=0.83; stroke: RR=0.91, p=0.72; target vessel revascularisation: RR=1.06, p=0.47) and overall clinical outcome (RR=0.90, p=0.22). There was also no difference in the rate of bleeding between PFT-guided and standard therapies (major bleeding: RR=0.97, p=0.78, minor bleeding: RR=0.89, p=0.19 and any bleeding: RR=1.04, p=0.33). CONCLUSION: Compared with standard DAPT with P2Y12 antagonists, using PFT to adjust antiplatelet therapy does not improve clinical outcomes. Therefore, the positions of key guidelines on routine testing in ACS should remain unchanged. In addition, the study highlights the need for well-designed and powered RCTs and standardised testing methodologies to provide reliable findings and definitive conclusions.

Antiplatelet therapy in cardiovascular disease: Current status and future directions.

Antiplatelet medications remain a cornerstone of therapy for atherosclerotic cardiovascular and cerebrovascular diseases. In primary prevention (patients with cardiovascular risk factors but no documented events, symptoms or angiographic disease), there is little evidence of benefit of any antiplatelet therapy, and such therapy carries the risk of excess bleeding. Where there is documented disease (secondary prevention), stable patients benefit from long-term antiplatelet monotherapy, aspirin being first choice in those with coronary heart disease and clopidogrel in those with cerebrovascular disease; moreover, recent evidence shows that low-dose rivaroxaban in combination with aspirin confers added benefit, in patients with stable cardiovascular and peripheral arterial disease. In patients with acute cerebrovascular disease, aspirin combined with clopidogrel reduces subsequent risk, while in acute coronary syndrome, dual antiplatelet therapy comprising aspirin and a P2Y12 inhibitor (clopidogrel, prasugrel or ticagrelor) confers greater protection than aspirin monotherapy, with prasugrel and ticagrelor offering greater antiplatelet efficacy with faster onset of action than clopidogrel. Although greater antiplatelet efficacy is advantageous in preventing thrombotic events, this must be tempered by increased risk of bleeding, which may be a particular issue in certain patient groups, as will be discussed. We will also discuss possible future approaches to personalisation of antiplatelet therapy.

Results of an international crowdsourcing survey on the treatment of non-ST segment elevation ACS patients at high-bleeding risk undergoing percutaneous intervention.

AIMS: Choosing an antiplatelet strategy in patients with non-ST segment elevation acute coronary syndrome (NSTE-ACS) at high bleeding risk (HBR), undergoing post-percutaneous coronary intervention (PCI), is complex. We used a unique open-source approach (crowdsourcing) to document if practices varied across a small, global cross-section of antiplatelet prescribers in the post-PCI setting. METHODS AND RESULTS: Five-hundred and fifty-nine professionals from 70 countries (the 'crowd') completed questionnaires containing single- or multi-option and free form questions regarding antiplatelet clinical practice in post-PCI NSTE-ACS patients at HBR. A threshold of 75% defined 'agreement'. There was strong agreement favouring monotherapy with either aspirin or a P2Y12 inhibitor following initial DAPT, within the first year (94%). No agreement was reached on the optimal duration of DAPT or choice of monotherapy: responses were in equipoise for shorter (≤3 months, 51%) or longer (≥6 months, 46%) duration, and monotherapy choice (45% aspirin; 53% P2Y12 inhibitor). Most respondents stated use of guideline-directed tools to assess risk, although clinical judgement was preferred by 32% for assessing bleeding risk and by 46% for thrombotic risk. CONCLUSION: The crowdsourcing methodology showed potential as a tool to assess current practice and variation on a global scale and to achieve a broad demographic representation. These preliminary results indicate a high degree of variation with respect to duration of DAPT, monotherapy drug of choice following DAPT and how thrombotic and bleeding risk are assessed. Further investigations should concentrate on interrogating practice variation between key demographic groups.

Netrin-1: Focus on its role in cardiovascular physiology and atherosclerosis.

The netrins form a family of laminin-related proteins which were first described as modulators of cell migration and axonal guidance during fetal development. Netrin-1 is the most extensively studied member of this family and, since its discovery, non-neural roles have been associated with it. Together with its receptors, DCC/neogenin and UNC5, netrin-1 has been shown to be involved in the regulation of angiogenesis, organogenesis, cancer and inflammation. An NF-κB-dependent truncated isoform of netrin-1 has also been shown to be produced in endothelial and some types of cancer cells, which both accumulates in and affects the function of the nucleus. In atherosclerosis, conflicting roles for netrin-1 have been reported on plaque progression via its receptor UNC5b. Whereas endothelial-derived netrin-1 inhibits chemotaxis of leukocytes and reduces the migration of monocytes to the atherosclerotic plaque, netrin-1 expressed by macrophages within the plaque plays a pro-atherogenic role, promoting cell survival, recruiting smooth muscle cells and inhibiting foam cell egress to the lymphatic system. In contrast, there is evidence that netrin-1 promotes macrophage differentiation to an alternative activated phenotype and induces expression of IL-4 and IL-13, while downregulate expression of IL-6 and COX-2. Further work is needed to elucidate the precise roles of the two isoforms of netrin-1 in different cell types in the context of atherosclerosis, and its potential as a putative novel therapeutic target in this disease.

ß-catenin promotes endothelial survival by regulating eNOS activity and flow-dependent anti-apoptotic gene expression.

Increased endothelial cell (EC) apoptosis is associated with the development of atherosclerotic plaques that develop predominantly at sites exposed to disturbed flow (DF). Strategies to promote EC survival may therefore represent a novel therapeutic approach in cardiovascular disease. Nitric oxide (NO) and ß-catenin have both been shown to promote cell survival and they interact in ECs as we previously demonstrated. Here we investigated the physiological role of ß-catenin as a mediator of NO-induced cell survival in ECs. We found that ß-catenin depleted human umbilical vein ECs (HUVEC) stimulated with pharmacological activators of endothelial NO synthase (eNOS) showed a reduction in eNOS phosphorylation (Ser1177) as well as reduced intracellular cyclic guanosine monophosphate levels compared to control cells in static cultures. In addition, ß-catenin depletion abrogated the protective effects of the NO donor, S-nitroso-N-acetylpenicillamine, during TNFα- and H2O2-induced apoptosis. Using an orbital shaker to generate shear stress, we confirmed eNOS and ß-catenin interaction in HUVEC exposed to undisturbed flow and DF and showed that ß-catenin depletion reduced eNOS phosphorylation. ß-catenin depletion promoted apoptosis exclusively in HUVEC exposed to DF as did inhibition of soluble guanylate cyclase (sGC) or ß-catenin transcriptional activity. The expression of the pro-survival genes, Bcl-2 and survivin was also reduced following inhibition of ß-catenin transcriptional activity, as was the expression of eNOS. In conclusion, our data demonstrate that ß-catenin is a positive regulator of eNOS activity and cell survival in human ECs. sGC activity and ß-catenin-dependent transcription of Bcl-2, survivin, BIRC3 and eNOS are essential to maintain cell survival in ECs under DF.

Changes in C-reactive protein in response to anti-inflammatory therapy as a predictor of cardiovascular outcomes: A systematic review and meta-analysis.

BACKGROUND: Despite the availability of aggressive lipid-lowering strategies, many patients remain at risk of cardiovascular events. C-reactive protein is a marker of inflammation elevated in patients at high risk of cardiovascular events. C-reactive protein has demonstrated value as a predictor of cardiovascular risk; however, it is unclear whether targeting C-reactive protein levels improves outcomes. This systematic review aimed to characterise the relationship between C-reactive protein and cardiovascular outcomes and to assess whether the magnitude of C-reactive protein reduction correlates to the extent of cardiovascular risk reduction. METHODS: A systematic review was conducted to identify randomised controlled trials that measured C-reactive protein before and after administration of therapies for cardiovascular disease and measured incidence of cardiovascular events. A meta-analysis of placebo-controlled studies assessed the relationship between extent of C-reactive protein reduction and cardiovascular risk reduction. Placebo-controlled studies where low-density lipoprotein and triglyceride data were available were also included in a meta-regression to assess the influence of these established risk factors on the efficacy of treatment when compared to C-reactive protein. RESULTS: Fifteen studies met the criteria for inclusion in this review, of which six were active comparator studies and nine were placebo controlled. Six placebo-controlled studies had data available for meta-regression. Eight studies demonstrated a reduction in events that could be explained by changes in lipid levels, whereas the results of five studies suggested that the association between C-reactive protein reduction and event rates cannot be explained by changes in lipid levels alone. No correlation was found between magnitude of C-reactive protein reduction and cardiovascular risk reduction. A strong correlation was found between C-reactive protein and low-density lipoprotein reduction (adjusted r 2 = 0.8). CONCLUSIONS: Targeting C-reactive protein does not offer additional benefit over targeting low-density lipoprotein across the general population in terms of cardiovascular risk reduction. However, there is value in targeting C-reactive protein in patients at high residual inflammatory risk despite non-elevated lipid levels or use of lipid-lowering therapy.

The effectiveness of a specialist Hypertension Clinic in reducing blood pressure: Factors determining blood pressure response.

OBJECTIVES: We retrospectively assessed both blood pressure response to management in a specialist Hypertension Clinic and the predictors of response. DESIGN: Retrospective data audit. SETTING: Hypertension Clinic at Guy's and St Thomas' Hospitals, London, UK. PARTICIPANTS: One hundred and twenty-two sequential patients attending the clinic between January 2017 and October 2018. Patients were excluded if they had previously been seen in the clinic, failed to attend their initial appointment or did not return after their initial appointment. OUTCOME MEASURE: Change in blood pressure readings between first and last recorded appointments. RESULTS: Systolic blood pressure decreased from 149 ± 20 mmHg to 140 ± 17 mmHg, and diastolic blood pressure from 92 ± 13 to 86 ± 1 mmHg, between the first and last recorded appointments (P < 0.05 for each). Those patients who reached target blood pressure were significantly younger than those who did not (age 45 ± 14 versus 52 ± 17 years, P = 0.0171); however, the degree of blood pressure reduction was independent of age, and younger subjects had a baseline blood pressure significantly lower than older subjects. Sex, body mass index and lifestyle interventions had an important effect on blood pressure reduction; however, these were not associated with attainment of target blood pressure. CONCLUSION: Specialist hypertension input is effective in helping patients to achieve target blood pressure, especially so in younger as compared to older hypertensive patients, but this appears to be related to the fact that blood pressure in younger patients is already closer to target at baseline. Blood pressure reduction is greater in women, those with higher body mass index and those who receive lifestyle interventions.

SNO-MLP (S-Nitrosylation of Muscle LIM Protein) Facilitates Myocardial Hypertrophy Through TLR3 (Toll-Like Receptor 3)-Mediated RIP3 (Receptor-Interacting Protein Kinase 3) and NLRP3 (NOD-Like Receptor Pyrin Domain Containing 3) Inflammasome Activation.

BACKGROUND: S-nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in the pathogenesis of cardiovascular disease. The aim of this study was to determine the role of SNO of MLP (muscle LIM protein) in myocardial hypertrophy, as well as the mechanism by which SNO-MLP modulates hypertrophic growth in response to pressure overload. METHODS: Myocardial samples from patients and animal models exhibiting myocardial hypertrophy were examined for SNO-MLP level using biotin-switch methods. SNO sites were further identified through liquid chromatography-tandem mass spectrometry. Denitrosylation of MLP by the mutation of nitrosylation sites or overexpression of S-nitrosoglutathione reductase was used to analyze the contribution of SNO-MLP in myocardial hypertrophy. Downstream effectors of SNO-MLP were screened through mass spectrometry and confirmed by coimmunoprecipitation. Recruitment of TLR3 (Toll-like receptor 3) by SNO-MLP in myocardial hypertrophy was examined in TLR3 small interfering RNA-transfected neonatal rat cardiomyocytes and in a TLR3 knockout mouse model. RESULTS: SNO-MLP level was significantly higher in hypertrophic myocardium from patients and in spontaneously hypertensive rats and mice subjected to transverse aortic constriction. The level of SNO-MLP also increased in angiotensin II- or phenylephrine-treated neonatal rat cardiomyocytes. S-nitrosylated site of MLP at cysteine 79 was identified by liquid chromatography-tandem mass spectrometry and confirmed in neonatal rat cardiomyocytes. Mutation of cysteine 79 significantly reduced hypertrophic growth in angiotensin II- or phenylephrine-treated neonatal rat cardiomyocytes and transverse aortic constriction mice. Reducing SNO-MLP level by overexpression of S-nitrosoglutathione reductase greatly attenuated myocardial hypertrophy. Mechanistically, SNO-MLP stimulated TLR3 binding to MLP in response to hypertrophic stimuli, and disrupted this interaction by downregulating TLR3-attenuated myocardial hypertrophy. SNO-MLP also increased the complex formation between TLR3 and RIP3 (receptor-interacting protein kinase 3). This interaction in turn induced NLRP3 (nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3) inflammasome activation, thereby promoting the development of myocardial hypertrophy. CONCLUSIONS: Our findings revealed a key role of SNO-MLP in myocardial hypertrophy and demonstrated TLR3-mediated RIP3 and NLRP3 inflammasome activation as the downstream signaling pathway, which may represent a therapeutic target for myocardial hypertrophy and heart failure.

The effect of aspirin on circulating netrin-1 levels in humans is dependent on the inflammatory status of the vascular endothelium.

In atherosclerotic animal models, the cyclo-oxygenase (COX)-inhibitor aspirin counteracts downregulation of endothelial-derived netrin-1, thus reducing arterial inflammation. We here explored the effect of aspirin on netrin-1 in healthy subjects undergoing influenza immunisation, which is an established experimental model of inflammation-related endothelial dysfunction. Our data showed that netrin-1 undergoes reduction (-29.25% from baseline; p=0.0017) in the presence of endothelial activation (VCAM-1 rose by 9.98% 2-days post-vaccination; p=0.0022). Aspirin counteracted vaccine-induced endothelial activation and reduction of netrin-1 in a dose-dependent manner (-3.06% and -17.03% from baseline at a dose of 300mg and 75mg respectively; p=0.0465 and p>0.05 vs untreated). Clopidogrel, which was used as a comparator due to its similar anti-platelet activity, also reduced endothelial activation but, unlike aspirin, enhanced netrin-1 levels (+20.96% from baseline; p=0.0033 vs untreated). A correlation analysis incorporating cytokines, hs-CRP, VCAM-1, TXB2 and PGE2, showed that changes in netrin-1 were directly related to PGE2 variations only (r=0.6103; p=0.0002). In a separate population of 40 healthy unimmunised volunteers, 28-day treatment with aspirin 300mg reduced netrin-1 (-18.76% from baseline; p=0.0012) without affecting endothelial markers or hs-CRP; as expected, aspirin suppressed TXB2 and PGE2. Netrin-1 and PGE2 levels were directly related (r=0.358; p=0.0015), but other parameters including TXB2, hs-CRP and endothelial markers, were not. In conclusion, aspirin counteracts downregulation of netrin-1 following endothelial dysfunction due to its anti-inflammatory effect on the activated endothelium. However, inhibition of COX-dependent prostanoids negatively modulates netrin-1 synthesis in healthy subjects, and this could give rise to aspirin-dependent reduction in netrin-1 under steady state conditions.

CXCR2 Inhibition - a novel approach to treating CoronAry heart DiseAse (CICADA): study protocol for a randomised controlled trial.

BACKGROUND: There is emerging evidence of the central role of neutrophils in both atherosclerotic plaque formation and rupture. Patients with lower neutrophil counts following acute coronary syndromes tend to have a greater coronary flow reserve, which is a strong predictor of long-term cardiovascular health. But so far, no data are available regarding the impact of neutrophil inhibition on cardiovascular clinical or surrogate endpoints. Therefore, the aim of this study is to investigate the effects of AZD5069, a cysteine-X-cysteine chemokine receptor 2 (CXCR2) inhibitor, on coronary flow reserve and coronary structure and function in patients with coronary artery disease. METHODS/DESIGN: Ninety subjects with coronary artery disease undergoing percutaneous coronary intervention will be included in this investigator-driven, randomised, placebo-controlled, double-blind, phase IIa, single-centre study. Participants will be randomised to receive either AZD5069 (40 mg) administered orally twice daily or placebo for 24 weeks. Change in coronary flow reserve as determined by 13N-ammonia positron emission tomography-computed tomography will be the primary outcome. Change in the inflammatory component of coronary plaque structure and the backward expansion wave, an invasive coronary physiological measure of diastolic function, will be assessed as secondary outcomes. DISCUSSION: Cardiovascular surrogate parameters, such as coronary flow reserve, may provide insights into the potential mechanisms of the cardiovascular effects of CXCR2 inhibitors. Currently, ongoing trials do not specifically focus on neutrophil function as a target of intervention, and we therefore believe that our study will contribute to a better understanding of the role of neutrophil-mediated inflammation in coronary artery disease. TRIAL REGISTRATION: EudraCT, 2016-000775-24 . Registered on 22 July 2016. International Standard Randomised Controlled Trial Number, ISRCTN48328178 . Registered on 25 February 2016.

Gender differences in cardiovascular prophylaxis: Focus on antiplatelet treatment.

Cardiovascular disease (CVD) represents the leading cause of death worldwide, and equally affects both sexes although women develop disease at an older age than men. A number of clinical evidence has identified the female sex as an independent factor for poor prognosis, with the rate of mortality and disability following an acute cardiovascular (CV) event being higher in women than men. It has been argued that the different level of platelet reactivity between sexes may account for a different responsiveness to anti-platelet therapy, with consequent important implications on clinical outcomes. However, conclusive evidence supporting the concept of a gender-dependent effectiveness of platelet inhibitors are lacking. On the contrary, sex-related dissimilarities have been evidenced in cardiovascular patients in terms of age of presentation, comorbidities such as obesity, diabetes and renal disease, and a different pharmacological approach to and effectiveness in controlling classical cardiovascular risk factors such as hypertension, glucose profile and lipid dysmetabolism. All these factors could place women at an increased level of cardiovascular risk compared to men, and may concur to an enhanced pro-thrombogenic profile. The purpose of this manuscript is to provide an overview of gender-related differences in cardiovascular treatment, in order to highlight the need to improve the pharmacological prophylaxis adopted in women through a more accurate evaluation of the overall cardiovascular risk profile with consequent establishment of a more effective and targeted anti-thrombotic strategy which is not limited to the use of anti-platelet agents.

Antiplatelet Therapy in Acute Coronary Syndrome.

Antiplatelet agents have for decades been used to improve outcomes in patients with acute coronary syndromes and have become increasingly valued, not only for their antithrombotic properties but also for their anti-inflammatory effects. The drug class continues to evolve as novel agents with increasingly efficacious antiplatelet actions are identified. This review will discuss antiplatelet agents, including aspirin, the P2Y12 receptor antagonists and the glycoprotein IIb/IIIa inhibitors, that are currently used to treat patients with unstable angina and myocardial infarction, focusing on their pharmacological properties and the clinical evidence supporting their use.