Novel mechanisms of thrombo-inflammation during infection: spotlight on neutrophil extracellular trap-mediated platelet activation.

A state-of-the-art lecture titled "novel mechanisms of thrombo-inflammation during infection" was presented at the ISTH Congress in 2022. Platelet, neutrophil, and endothelial cell activation coordinate the development, progression, and resolution of thrombo-inflammatory events during infection. Activated platelets and neutrophil extracellular traps (NETs) are frequently observed in patients with sepsis and COVID-19, and high levels of NET-derived damage-associated molecular patterns (DAMPs) correlate with thrombotic complications. NET-associated DAMPs induce direct and indirect platelet activation, which in return potentiates neutrophil activation and NET formation. These coordinated interactions involve multiple receptors and signaling pathways contributing to vascular and organ damage exacerbating disease severity. This state-of-the-art review describes the main mechanisms by which platelets support NETosis and the key mechanisms by which NET-derived DAMPs trigger platelet activation and the formation of procoagulant platelets leading to thrombosis. We report how these DAMPs act through multiple receptors and signaling pathways differentially regulating cell activation and disease outcome, focusing on histones and S100A8/A9 and their contribution to the pathogenesis of sepsis and COVID-19. We further discuss the complexity of platelet activation during NETosis and the potential benefit of targeting selective or multiple NET-associated DAMPs to limit thrombo-inflammation during infection. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

S100A8/A9 drives the formation of procoagulant platelets through GPIbα.

S100A8/A9, also known as "calprotectin" or "MRP8/14," is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Preferential uptake of SARS-CoV-2 by pericytes potentiates vascular damage and permeability in an organoid model of the microvasculature.

AIMS: Thrombotic complications and vasculopathy have been extensively associated with severe COVID-19 infection; however, the mechanisms inducing endotheliitis and the disruption of endothelial integrity in the microcirculation are poorly understood. We hypothesized that within the vessel wall, pericytes preferentially take up viral particles and mediate the subsequent loss of vascular integrity. METHODS AND RESULTS: Immunofluorescence of post-mortem patient sections was used to assess pathophysiological aspects of COVID-19 infection. The effects of COVID-19 on the microvasculature were assessed using a vascular organoid model exposed to live viral particles or recombinant viral antigens. We find increased expression of the viral entry receptor angiotensin-converting enzyme 2 on pericytes when compared to vascular endothelium and a reduction in the expression of the junctional protein CD144, as well as increased cell death, upon treatment with both live virus and/or viral antigens. We observe a dysregulation of genes implicated in vascular permeability, including Notch receptor 3, angiopoietin-2, and TEK. Activation of vascular organoids with interleukin-1ß did not have an additive effect on vascular permeability. Spike antigen was detected in some patients' lung pericytes, which was associated with a decrease in CD144 expression and increased platelet recruitment and von Willebrand factor (VWF) deposition in the capillaries of these patients, with thrombi in large vessels rich in VWF and fibrin. CONCLUSION: Together, our data indicate that direct viral exposure to the microvasculature modelled by organoid infection and viral antigen treatment results in pericyte infection, detachment, damage, and cell death, disrupting pericyte-endothelial cell crosstalk and increasing microvascular endothelial permeability, which can promote thrombotic and bleeding complications in the microcirculation.

Five-year follow-up of intracoronary autologous cell therapy in acute myocardial infarction: the REGENERATE-AMI trial.

AIMS: The long-term outcomes of the intracoronary delivery of autologous bone marrow-derived cells (BMCs) after acute myocardial infarction are not well established. Following the promising 1 year results of the REGENERATE-AMI trial (despite it not achieving its primary endpoint), this paper presents the analysis of the 5 year clinical outcomes of these acute myocardial infarction patients who were treated with an early intracoronary autologous BMC infusion or placebo. METHODS AND RESULTS: A 5 year follow-up of major adverse cardiac events (defined as the composite of all-cause death, recurrent myocardial infarction, and all coronary revascularization) and of rehospitalization for heart failure was completed in 85 patients (BMC n = 46 and placebo n = 39). The incidence of major adverse cardiac events was similar between the BMC-treated patients and the placebo group (26.1% vs. 18.0%, P = 0.41). There were no cases of cardiac death in either group, but an increase in non-cardiac death was seen in the BMC group (6.5% vs. 0%, P = 0.11). The rates of recurrent myocardial infarction and repeat revascularization were similar between the two groups. There were no cases of rehospitalization for heart failure in either group. CONCLUSION: This 5 year follow-up analysis of the REGENERATE-AMI trial did not show an improvement in clinical outcomes for patients treated with cell therapy. This contrasts with the 1 year results which showed improvements in the surrogate outcome measures of ejection fraction and myocardial salvage index.

Galectin-9 activates platelet ITAM receptors glycoprotein VI and C-type lectin-like receptor-2.

BACKGROUND: Platelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate-binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. OBJECTIVE: In this study, we investigated the role of galectin-9 (Gal-9) as a novel ligand for platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2). METHODS: Platelet spreading, aggregation, and P-selectin expression in response to Gal-9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid-phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal-9 and GPVI, and immunoprecipitation for detecting CLEC-2 phosphorylation. Wild-type (WT), GPVI-knockout (Gp6-/- ), and GPVI and CLEC-2-double knockout (Gp6-/- /Gp1ba-Cre-Clec1bfl/fl ) mice were used. RESULTS: We have shown that recombinant Gal-9 stimulates aggregation in human and mouse washed platelets dose-dependently. Platelets from both species adhere and spread on immobilized Gal-9 and express P-selectin. Gal-9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins known to lie downstream of GPVI and CLEC-2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI-deficient murine platelets exhibited significantly impaired aggregation in response to Gal-9, which was further abrogated in GPVI and CLEC-2-double-deficient platelets. CONCLUSIONS: We have identified Gal-9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC-2.

CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis.

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.

Umbilical cord-derived mesenchymal stromal cells in cardiovascular disease: review of preclinical and clinical data.

The human umbilical cord has recently emerged as an attractive potential source of mesenchymal stromal cells (MSCs) to be adopted for use in regenerative medicine. Umbilical cord MSCs (UC-MSCs) not only share the same features of all MSCs such as multi-lineage differentiation, paracrine functions and immunomodulatory properties, they also have additional advantages, such as no need for bone marrow aspiration and higher self-renewal capacities. They can be isolated from various compartments of the umbilical cord (UC) and can be used for autologous or allogeneic purposes. In the past decade, they have been adopted in cardiovascular disease and have shown promising results mainly due to their pro-angiogenic and anti-inflammatory properties. This review offers an overview of the biological properties of UC-MSCs describing available pre-clinical and clinical data with respect to their potential therapeutic use in cardiovascular regeneration, with current challenges and future directions discussed.

Circulatory support and stem cell therapy in the management of advanced heart failure: a concise review of available evidence.

Stem cell therapy utilizing bone marrow mononuclear cells (BMC's) is a potential strategy to treat heart failure patients with improvement in symptom profile and cardiac function. We describe a rationale for concurrent BMC and left ventricular assist device therapy in selected heart failure patients. This combination therapy has demonstrated improved myocardial perfusion and cardiac function in patients with advanced ischemic cardiomyopathy. Moreover, preclinical data support improved cell retention with left ventricular unloading. The beneficial effects of BMC's are likely through a paracrine mechanism initiating a 'cardiac-repair' process. Combination therapy of BMC's and a left ventricular assist device may exhibit a synergistic effect with improved engraftment of BMC's through left ventricular unloading.

The Impact of Cell Therapy on Cardiovascular Outcomes in Patients With Refractory Angina.

RATIONALE: Cell-based therapies are a novel potential treatment for refractory angina and have been found to improve markers of angina. However, the effects on mortality and major adverse cardiac events (MACE) have not been definitively investigated. OBJECTIVE: To investigate the efficacy and safety of stem cell treatment compared with optimal medical treatment for refractory angina by conducting an updated meta-analysis, looking at clinical outcomes. METHODS AND RESULTS: We performed a systematic review and meta-analysis of randomized controlled trials using the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. A comprehensive search was performed of PubMed, EMBASE (Excerpta Medica database), Cochrane, ClinicalTrials.gov , Google Scholar databases of randomized controlled trials, and scientific session abstracts. Studies were deemed eligible if they met the following criteria: (1) full-length publications in peer-reviewed journals; (2) evaluated cell therapy use in patients with no further revascularisation options while on optimal medical treatment; (3) patients had ongoing angina, Canadian Cardiovascular Society class II-IV; and (4) included a placebo/control arm. We calculated risk ratios for all-cause mortality, combined MACE events. We assessed heterogeneity using χ2 and I2 tests. We identified 1191 citations with 8 randomized controlled trials meeting inclusion criteria involving 526 patients. Outcomes pooled were MACE, mortality, and indices of angina (angina episodes, Canadian Cardiovascular Society angina class, exercise tolerance, and antianginal medications). Our analysis showed a decreased risk of both MACE (odds ratio, 0.41; CI, 0.25-0.70) and mortality (odds ratio, 0.24; 95% CI, 0.10-0.60) in cell-treated patients compared with patients on maximal medical therapy. This was supported by improvements in surrogate end points of anginal episodes, use of antianginal medications, Canadian Cardiovascular Society class, and exercise tolerance. CONCLUSIONS: In addition to improvements in indices of angina, cell-based therapies improve cardiovascular outcomes (mortality/MACE) in patients with refractory angina. Given the premature termination of the phase III study, this supports the need for further definitive trials. Prospero Registration : URL: https://www.crd.york.ac.uk/prospero/ . Unique identifier: CRD42018084257.

Risk scoring to guide antiplatelet therapy post-percutaneous coronary intervention for acute coronary syndrome results in improved clinical outcomes.

Aims: To use the Global Registry of Acute Coronary Events (GRACE) and Can Rapid risk stratification of Unstable angina patients Suppress ADverse outcomes with Early implementation of the ACC/AHA guidelines (CRUSADE) scores to risk stratify antiplatelet treatment post-acute coronary syndrome (ACS). Methods and results: This was a prospective registry of 3374 patients undergoing percutaneous coronary intervention for ACS between 2013 and 2015 at a UK cardiac centre. Patients with either low GRACE or high CRUSADE risk scores were stratified either to clopidogrel therapy or ticagrelor was used. The primary endpoint was major adverse cardiac events (MACE) defined as death, non-fatal myocardial infarction, stroke, or target vessel revascularization with bleeding rates as a secondary outcome, assessed at a median follow-up of 1.8 years (interquartile range 0.8-3.4 years). A total of 1723 (51.1%) patients were risk stratified to either clopidogrel (n = 520) or ticagrelor treatment (n = 1203), with the remaining 1651 not risk scored and treated with clopidogrel therapy. Patients in the risk score stratified group were older than the control group otherwise the groups were similar. Over the follow-up period, a significant reduction in MACE rates between the patients' risk score stratified and control (clopidogrel therapy) (13.7% vs. 19.7%, P < 0.0001) was seen [hazard ratio (HR) 0.61, 95% confidence interval (CI) 0.31-0.86]. This persisted after adjusting for baseline variables (HR 0.65, 95% CI 0.37-0.89) and propensity matching (HR = 0.63, 95% CI 0.27-0.93; P = 0.0015) No significant differences in the rate of major bleeding were seen between the groups (5.3% vs. 5.1%, P = 0.86). In the risk-stratified group, no difference in outcome (ischaemic/bleeding) was seen between clopidogrel and ticagrelor. Conclusion: Our registry data suggest that using appropriate risk scoring to guide antiplatelet therapy after ACS is safe and can result in improved clinical outcomes.

Dual-site right ventricular pacing in patients undergoing cardiac resynchronization therapy: Results of a multicenter propensity-matched analysis.

BACKGROUND: Dual-site right ventricular pacing (Dual RV) has been proposed as an alternative for patients with heart failure undergoing cardiac resynchronization therapy (CRT) with a failure to deliver a coronary sinus (CS) lead. Only short-term hemodynamic and echocardiographic results of Dual RV are available. We aimed to assess the long-term results of Dual RV and its impact on survival. METHODS: Multicenter retrospective assessment of all CRT implants during a 12-year period. Patients with failed CS lead implantation, treated with Dual RV, were followed and assessed for the primary endpoint of all-cause mortality and/or heart transplant. A control group was obtained from contemporary patients using propensity matching for all available baseline variables. RESULTS: Ninety-three patients were implanted with Dual RV devices and compared with 93 matched controls. During a median of 1,273 days (interquartile range 557-2,218), intention-to-treat analysis showed that all-cause mortality and/or heart transplant was higher in the Dual RV group (adjusted hazard ratio [HR] = 1.66, 95% confidence interval [CI] 1.12-2.47, P = 0.012). As-treated analysis yielded similar results (HR = 1.97, 95% CI 1.31-2.96, P = 0.001). Cardiac device-related infections occurred seven times more frequently in the Dual RV site group (HR = 7.60, 95% CI 1.51-38.33, P = 0.014). Among Dual RV nonresponders, four had their apical leads switched off, five required an epicardial LV lead insertion, a transseptal LV lead was implanted in two, and in nine patients, after reviewing the CS venogram, a new CS lead insertion was successfully attempted. CONCLUSION: Dual RV pacing is associated with worse clinical outcomes and higher complication rates than conventional CRT.

Molecular basis of thyrotropin and thyroid hormone action during implantation and early development.

BACKGROUND: Implantation and early embryo development are finely regulated processes in which several molecules are involved. Evidence that thyroid hormones (TH: T4 and T3) might be part of this machinery is emerging. An increased demand for TH occurs during gestation, and any alteration in maternal thyroid physiology has significant implications for both maternal and fetal health. Not only overt but also subclinical hypothyroidism is associated with infertility as well as with obstetric complications, including disruptions and disorders of pregnancy, labor, delivery, and troubles in early neonatal life. METHODS: We searched the PubMed and Google Scholar databases for articles related to TH action on ovary, endometrium, trophoblast maturation and embryo implantation. In addition, articles on the regulation of TH activity at cellular level have been reviewed. The findings are hereby summarized and critically discussed. RESULTS: TH have been shown to influence endometrial, ovarian and placental physiology. TH receptors (TR) and thyrotropin (thyroid-stimulating hormone: TSH) receptors (TSHR) are widely expressed in the feto-maternal unit during implantation, and both the endometrium and the trophoblast might be influenced by TH either directly or through TH effects on the synthesis and activity of implantation-mediating molecules. Interestingly, due to the multiplicity of mechanisms involved in TH action (e.g. differential expression of TR isoforms, heterodimeric receptor partners, interacting cellular proteins, and regulating enzymes), the TH concentration in blood is not always predictive of their cellular availability and activity at both genomic and nongenomic level. CONCLUSIONS: In addition to the known role of TH on the hormonal milieu of the ovarian follicle cycle, which is essential for a woman's fertility, evidence is emerging on the importance of TH signaling during implantation and early pregnancy. Based on recent observations, a local action of TH on female reproductive organs and the embryo during implantation appears to be crucial for a successful pregnancy. Furthermore, an imbalance in the spatio-temporal expression of factors involved in TH activity might induce early arrest of pregnancy in women considered as euthyroid, based on their hormonal blood concentration. In conclusion, alterations of the highly regulated local activity of TH may play a crucial, previously underestimated, role in early pregnancy and pregnancy loss. Further studies elucidating this topic should be encouraged.

Sella turcica atypical teratoid/rhabdoid tumor complicated with lung metastasis in an adult female.

Here we present the case of a 60-year-old woman with a rare sellar region atypical teratoid/rhabdoid tumor (AT/RT), complicated by lung metastasis and treated with neurosurgery, radiotherapy, and chemotherapy. The patient had recurrent headache associated with left cavernous sinus syndrome after a previous endonasal transsphenoidal resection for a presumptive pituitary macroadenoma. Pituitary magnetic resonance imaging showed a tumor regrowth in the original location with a haemorrhagic component involving the left cavernous sinus. A near complete transsphenoidal resection of the sellar mass was performed followed by 3 months of stereotactic radiotherapy. Because of a worsening of the general clinical conditions, respiratory failure, and asthenia, the patient underwent a contrast enhanced computer tomography of the whole body which showed the presence of lung metastasis. The histopathological diagnosis on samples from pituitary and lung tissues was AT/RT. The patient survived 30 months after diagnosis regardless chemotherapy. In the adult, the AT/RT should be considered as a possible rare, aggressive, and malignant neoplasm localized in the sella turcica.