Thrombus Samples Research Articles

Microbial signatures in chronic thromboembolic pulmonary hypertension thrombi: Insights from metagenomic profiling of fresh and organized thrombi

ObjectiveMany studies have reported microbial signatures in thrombi at major vascular sites, such as the coronary artery and the middle cerebral artery, which are critical for maintaining proper blood flow and oxygenation. Chronic thromboembolic pulmonary hypertension (CTEPH) is a condition involving non-resolving thrombosis that has not been fully studied. This study explored the microbial taxonomy and functional profiles of both fresh and organized thrombi associated with CTEPH to investigate the role of microbiota in thrombus non-resolving. MethodsIn this study, 12 CTEPH fresh thrombi and 12 organized thrombi were collected from 14 patients with CTEPH. Metagenomic sequencing was employed to explore the genomic information of all microorganisms in the thrombus samples. ResultsOur data demonstrated a diverse range of microorganisms in CTEPH thrombi, whether fresh or organized. Notably, a considerable proportion (54.7 %) of sequencing data could not be classified into the relative microbial taxa, highlighting the complexity and novelty of the thrombus ecosystem. Although there were no significant differences in microbial community structure between the two groups, the abundance of dominant microbial species varied. Leuconostoc sp. DORA 2, Staphylococcus aureus, and Aliidongia dinghuensis were common dominant species in CTEPH thrombus. Organized thrombus significantly increased the relative abundance of Staphylococcus aureus, which was confirmed to effectively distinguish between organized and fresh thrombi by LeFSe analysis and random forest analysis. Functional annotation using both the KEGG and eggNOG databases revealed that organized thrombi exhibit stronger metabolic functions, particularly in amino acid metabolism. ConclusionsOur findings suggest that microbial composition and function may play an important role in thrombus organization. Targeting inflammation to prevent thrombosis presents promising opportunities for further research in this area.

Relationship between thrombus vWF and NETs with clinical severity and peripheral blood immunocytes' indicators in patients with acute ischemic stroke.

To investigate the association between von Willebrand factor (vWF) and neutrophil extracellular traps (NETs) in thrombus with clinical severity and peripheral blood immunocytes' indicators in patients with early-stage acute ischemic stroke (AIS). A retrospective study was conducted using the clinical data of 66 patients with AIS who underwent endovascular mechanical thrombectomy and had their thrombus samples collected. The concentrations of vWF and NETs in the thrombus samples were quantitatively assessed. Peripheral blood samples taken in the early stages of the disease were analyzed for total white blood cell counts (WBC), ratios of neutrophils (NEU%), lymphocytes (LYM%), eosinophils (EOS%), and monocytes (MONO%). The severity of clinical symptoms in these patients was evaluated using the modified Rankin Scale (mRS), Essen Stroke Risk Score (ESRS), Barthel Index (BI), and National Institute of Health Stroke Scale (NIHSS). Higher vWF levels in thrombus were associated with lower NIHSS scores, while higher NETs levels were associated with higher initial NIHSS scores. In the early stages of AIS, WBC count and vWF levels were negatively correlated, as well as NEU%. LYM% was positively correlated with vWF level; however, it was negatively correlated with NETs. EOS% was positively correlated with vWF levels. In the early stages of AIS, a higher peripheral WBC count and NEU%, combined with decreased EOS% and LYM%, were significantly correlated with a lower vWF level in the thrombus, potentially indicating more severe symptoms. Consequently, the timely administration of vWF-targeted medications is recommended for such patients. Reduced LYM% is indicative of elevated NETs levels and correlated with more severe clinical symptoms. Therefore, the prompt initiation of NETs-targeted medication is warranted for these patients.

Abstract 1069: Novel Approaches Dissecting Human Thrombus Composition

Introduction: Thrombus formation is a key contributor to ischemic stroke and cardiovascular diseases. Variations in thrombus composition involving platelets, fibrin, and leukocytes play a pivotal role in the pathophysiology, impacting clinical outcomes. Understanding its character and the interplay with Neutrophil Extracellular Traps (NETs) is clinically vital due to treatment response and outcome. This study introduces light-sheet microscopy and holotomography to dissect clot composition, aiming to enhance clinical insights into thrombosis. Unraveling these complexities holds promise for targeted interventions and improved patient outcomes. Aims: This study aimed to enhance our understanding of human blood thrombus composition by employing a novel approach using light-sheet microscopy and holotomography. Methods: Human thrombus samples were obtained by mechanical thrombectomy from acute ischemic stroke patients. To access the thrombus structure, the samples were cleared by a tissue-clearing rapid system, and light-sheet microscope imaging was obtained with CitH3, CD45, P-selectin, and fibrin. Holotomography images were obtained from cryosectioned thrombus at 5μm thickness and stained with DAPI and CitH3. Holotomography provided quantitative information on the refractive index of thrombus components. Comparative analysis with traditional imaging methods was performed to assess the advantages of the novel approaches. Results: The results revealed a comprehensive visualization of the spatial arrangement of human thrombus composition through light-sheet microscopy imaging. Holotomography successfully acquired a three-dimensional image of a thrombus section sample. HT images were used to qualitatively distinguish differences in neutrophil RI distribution by NETosis and differences in RI values within the immune cell population. Conclusion: This study demonstrates the efficacy of combining light sheet microscopy and holotomography to comprehensively dissect human thrombus composition. The integration of these advanced imaging techniques provides an understanding of thrombosis, surpassing the limitations of traditional methods.

Validation of large animal models in mechanical valve research: a histologic comparison.

Mechanical valves still require life-long anticoagulation. Preclinical animal testing is a crucial step in the assessment of valves; however, the chosen animal model should be carefully considered, and a well-controlled animal model of mechanical valve thrombosis has not been established yet. In this study, a histopathologic comparison was performed to evaluate the representativity of pigs and sheep as large animal models in bileaflet mechanical valve thrombosis research. 10 pigs and 8 sheep were implanted with a bileaflet mechanical valve in pulmonary position. During follow-up, no anticoagulative therapy was administered. Pigs were sacrificed between 14 and 38 days for explantation and assessment of the valve. Sheep were sacrificed between 71 and 155 days. Thrombus samples were processed and (immuno)histochemical stainings were applied. A pathologist evaluated the samples morphologically and semiquantitatively and compared these samples to available slides from 3 human patients who underwent redo surgery for acute bileaflet mechanical valve thrombosis, caused by insufficient anticoagulation. All pigs showed macroscopically evident thrombi on the mechanical valve surface at sacrifice. In contrast, none of the sheep showed any sign of thrombus formation. Histology showed a high fibrin content in thrombi of both human and porcine cases (3/3 vs 8/10). Porcine thrombi showed more cellular organization (0/3 vs 6/10), more calcification (0/3 vs 9/10) and more endothelialization (0/3 vs 6/10). Inflammatory cells were present in all samples and were considered physiological. Contrary to sheep, pigs develop thrombi on their mechanical valves in the short-term if no anticoagulation is administered. Histologic comparison of human and porcine thrombi shows comparable findings. The pig model might serve interestingly for further research on valve thrombosis, if it shows not to be an overly aggressive model.

Multimodal detection and analysis of microplastics in human thrombi from multiple anatomically distinct sites

Microplastic (MP) pollution has emerged as a significant environmental concern worldwide. While extensive research has focused on their presence in marine organisms and ecosystems, their potential impact on human health, particularly on the circulatory system, remains understudied. This project aimed to identify and quantify the mass concentrations, polymer types, and physical properties of MPs in human thrombi surgically retrieved from both arterial and venous systems at three anatomically distinct sites, namely, cerebral arteries in the brain, coronary arteries in the heart, and deep veins in the lower extremities. Furthermore, this study aimed to investigate the potential association between the levels of MPs and disease severity. Thrombus samples were collected from 30 patients who underwent thrombectomy procedures due to ischaemic stroke (IS), myocardial infarction (MI), or deep vein thrombosis (DVT). Pyrolysis-gas chromatography mass spectrometry (Py-GC/MS) was employed to identify and quantify the mass concentrations of the MPs. Laser direct infrared (LDIR) spectroscopy and scanning electron microscopy (SEM) were used to analyse the physical properties of the MPs. Demographic and clinical information were also examined. A rigorous quality control system was used to eliminate potential environmental contamination. MPs were detected by Py-GC/MS in 80% (24/30) of the thrombi obtained from patients with IS, MI, or DVT, with median concentrations of 61.75μg/g, 141.80μg/g, and 69.62μg/g, respectively. Among the 10 target types of MP polymers, polyamide 66 (PA66), polyvinyl chloride (PVC), and polyethylene (PE) were identified. Further analyses suggested that higher concentrations of MPs may be associated with greater disease severity (adjusted β=7.72, 95% CI: 2.01-13.43, p<0.05). The level of D-dimer in the MP-detected group was significantly higher than that in the MP-undetected group (8.3±1.5μg/L vs 6.6±0.5μg/L, p<0.001). Additionally, LDIR analysis showed that PE was dominant among the 15 types of identified MPs, accounting for 53.6% of all MPs, with a mean diameter of 35.6μm. The shapes of the polymers detected using LDIR and SEM were found to be heterogeneous. This study presents both qualitative and quantitative evidence of the presence of MPs, and their mass concentrations, polymer types, and physical properties in thrombotic diseases through the use of multimodal detection methods. Higher concentrations of MPs may be associated with increased disease severity. Future research with a larger sample size is urgently needed to identify the sources of exposure and validate the observed trends in the study. This study was funded by the SUMC Scientific Research Initiation Grant (SRIG, No. 009-510858038), Postdoctoral Research Initiation Grant (No. 202205230031-3), and the 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (No. 2020LKSFG02C).

Risk factors, susceptibility vessel sign and thrombosis pathology for prognosis inpatients with different subtypes of acute ischemic stroke treated with mechanical thrombectomy

Abstract Purpose This study highlights the relationship between stroke subtypes, thrombosis pathology, and SVS and identifies probable risk factors affecting recanalization and outcomes in endovascular treatment. Methods A prospective study was conducted in which 53 AIS patients received intravenous thrombolysis and mechanical thrombectomy (MT) from January 2017 to December 2019 in a native Hospital. The recanalization was evaluated using modified cerebral infarction thrombolysis grade and prognosis at 3 months using modified rankin Scale (mRS). The risk factors were identified using univariate and multivariate logistic regression analyses. The thrombus samples were stained to determine their composition. Magnetic SWI was used to determine SVS. Results Age, locations of occlusion, and treatments were significantly different of TOAST subtypes (P &amp;lt; 0.05). The time from door to puncture (TDP) and time of operation (TO) were significantly lower in the successful recanalization group (P &amp;lt; 0.05). National Institutes of Health Stroke Scale (NIHSS), MT, TDP, time from onset to recanalization (TOR), and TO have significant differences between the two groups of mRS ≤ 2 and &amp;gt; 3. The occurrence of symptomatic intracranial hemorrhage (sICH) significantly affected the mRS score. The red and mixed thrombi were common in patients with positive SVS whereas the white thrombi were associated with the negative SVS. Conclusion Improved recanalization and prognosis was observed in early MT and when endovascular operations time was reduced.

Detecting prokaryote-specific gene and other bacterial signatures in thrombi from patients with acute ischemic stroke

Background and purposeMicrobial infection has been associated with thrombogenesis. This study aimed to detect bacterium-specific genes and other signatures in thrombi from patients with acute ischemic stroke and to relate these signatures to clinical characteristics.MethodsBlood samples were collected before thrombectomy procedures, and thrombus samples were obtained during the procedure. Identification and classification of bacteria in the samples were accomplished using 16 S rRNA gene sequencing. Bacterium-specific structures were observed with transmission electron microscopy. Bacterium-specific biomarkers were detected through immunohistochemical staining.Results16 S rRNA gene was detected in 32.1% of the thrombus samples from 81 patients. Bacillus (0.04% vs. 0.00046%, p = 0.003), Parabacteroides (0.20% vs. 0.09%, p = 0.029), Prevotella (1.57% vs. 0.38%, p = 0.010), Streptococcus (1.53% vs. 0.29%, p = 0.001), Romboutsia (0.18% vs. 0.0070%, p = 0.029), Corynebacterium (1.61% vs. 1.26%, p = 0.026) and Roseburia (0.53% vs. 0.05%, p = 0.005) exhibited significantly higher abundance in thrombi compared to arterial blood. Bacteria-like structures were observed in 22 (27.1%), while whole bacteria-like structures were observed in 7 (8.6%) thrombi under transmission electron microscopy. Immunohistochemical staining detected bacterium-specific monocyte/macrophage markers in 51 (63.0%) out of 81 thrombi. Logistic regression analysis indicated that alcohol consumption was associated with a higher bacteria burden in thrombi (odds ratio = 3.19; 95% CI, 1.10–9.27; p = 0.033).ConclusionBacterial signatures usually found in the oral cavity and digestive tract were detected in thrombi from patients with ischemic stroke. This suggests a potential involvement of bacterial infection in the development of thrombosis. Long-term alcohol consumption may potentially enhance this possibility.

Таксономическая характеристика бактериальной ДНК, выделенной из тромбов пациентов с ишемическим инсультом

There is evidence of the role of microorganisms in endothelial damage and, as a consequence of activation of blood coagulation pathways, thrombosis, which in turn is one of the risk factors for the development of acute cerebrovascular accidents (ACVA). Objective. To analyze the microbiological profile of DNA isolated from thrombus of patients with ACVA. Materials and methods. Fifty-seven thrombus samples obtained lifetime from the vessels of patients with ischemic-type ACVA during thrombectomy and thrombaspiration were selected for the study. All patients with ACVA were in the period of coronavirus infection reconvalescence. Twelve blood samples from patients without infectious diseases were used for comparison. Amplification of DNA isolated from biological material was performed using primers specific to the 16S rRNA gene fragment. The obtained PCR products were sequenced by Sanger sequencing method, with subsequent processing of chromatograms in the BCV package. RDP Classifier was used to determine the taxonomic affiliation of DNA sequences. Statistical analysis was performed in the R environment using Pearson's and Fisher's χ2 criteria. Test results were considered significant at p &lt; 0.05. Results. Using the RDP Classifier, the taxonomic affiliation of 16S rRNA gene fragment amplification products were determined for 43 blood and clot samples. DNA of Actinobacteria and Firmicutes taxa were most represented in the clot samples of patients with ACVA. Of the Firmicutes and Proteobacteria taxa, Firmicutes predominated in thrombi, whereas Proteobacteria predominated in blood. The obtained data are consistent with the data on the key role of systemic intestinal inflammation in the pathogenesis of ACVA. Conclusion. DNA fragments of Actinomycetales, Bacillales, and Clostridiales bacteria found in thrombi of patients with ACVA indicate that they can penetrate the damaged epithelial barrier and promote platelet aggregation and thrombus formation, increasing the risk of ischemic stroke. Key words: ischemic stroke, acute cerebral circulatory disorders, bacterial infection, RDP Classifier

Atherosclerotic Autoantigen ALDH4A1 as a Novel Immunological Indicator for Plaque Erosion in Patients with ST Segment Elevated Myocardial Infarction.

Aldehyde dehydrogenase 4A1 (ALDH4A1) was recently reported to be a novel autoantigen of atherosclerosis. However, its role in different phenotypes of acute coronary syndrome remains unclear. Herein, we planned to explore the circulating and regional expression of ALDH4A1 in patients with plaque rupture (PR) and plaque erosion (PE) determined by optical coherence tomography (OCT). After applying the inclusion and exclusion criteria, a prospective series of 312 patients with ST segment elevated myocardial infarction (STEMI), including 161 patients with PR and 151 patients with PE determined by OCT, were enrolled for plasma ALDH4A1 testing. In addition, ALDH4A1 was quantified using immunofluorescence in aspirated coronary thrombus samples obtained from 31 patients with PR and 25 patients with PE. In addition, we established an atherosclerosis mouse model and analyzed the distribution of ALDH4A1 expression in different mouse organs. Furthermore, we compared the level of ALDH4A1 in the spleen and carotid artery between Apoe-/- and C57 mice. The results showed that the plasma level of ALDH4A1 was significantly higher in STEMI patients with PE than in those with PR (4.6 ng/mL [2.2-8.7] vs. 3.5 ng/mL [1.6-5.6] p = 0.005). The expression of ALDH4A1 in aspirated coronary thrombi was also significantly higher in patients with PE than in those with PR (mean gray value: 32.0 [23.6-40.6] vs. 16.8 [14.0-24.5], p < 0.001). In animal models, the expression of ALDH4A1 is much higher in the spleen than in other organs, and the level of ALDH4A1 is significantly elevated in the spleen and carotid artery of Apoe-/- mice compared with C57 mice. The high levels of ALDH4A1 in the plasma and aspirated coronary thrombi independently correlated with PE in patients with STEMI. These results suggested that ALDH4A1 is involved in the mechanism of PE and serves as a promising biomarker and treatment target for patients with PE.

Integrated analysis of single-cell and bulk RNA sequencing data reveals an immunostimulatory microenvironment in tumor thrombus of osteosarcoma

Tumor thrombus of bone sarcomas represents a unique reservoir for various types of cancer and immune cells, however, the investigation of tumor thrombus at a single-cell level is very limited. And it is still an open question to identify the thrombus-specific tumor microenvironment that is associated with the tumor-adaptive immune response. Here, by analyzing bulk tissue and single-cell level transcriptome from the paired thrombus and primary tumor samples of osteosarcoma (OS) patients, we define the immunostimulatory microenvironment in tumor thrombus of OS with a higher proportion of tumor-associated macrophages with M1-like states (TAM-M1) and TAM-M1 with high expression of CCL4. OS tumor thrombus is found to have upregulated IFN-γ and TGF-β signalings that are related to immune surveillance of circulating tumor cells in blood circulation. Further multiplexed immunofluorescence staining of the CD3/CD4/CD8A/CD68/CCL4 markers validates the immune-activated state in the tumor thrombus samples. Our study first reports the transcriptome differences at a single-cell level between tumor thrombus and primary tumor in sarcoma.

Thrombus Signal on T1-Weighted Black-Blood MR Predicts Outcomes of Catheter-Directed Thrombolysis in Acute Deep Vein Thrombosis.

Catheter-directed thrombolysis (CDT) is an effective therapy for acute deep vein thrombosis (DVT). However, predicting the CDT outcomes remains elusive. We hypothesized that the thrombus signal on T1-weighted black-blood magnetic resonance (MR) can provide insight into CDT outcomes in acute DVT patients. A total of 117 patients with acute iliofemoral DVT were enrolled for T1-weighted black-blood MR before CDT in this prospective study. Based on the signal contrast between thrombus and adjacent muscle, patients were categorized into the iso-intense thrombus (Iso-IT), hyper-intense thrombus (Hyper-IT), and mixed iso-/hyper-intense thrombi (Mixed-IT) groups. Immediate treatment outcome (i.e., vein patency) and long-term treatment outcome (i.e., the incidence rate of postthrombotic syndrome) were accessed by the same expert. Histological analysis and iron quantification were performed on thrombus samples to characterize the content of fibrin, collagen, and the ratio of Fe3+ to total iron. Compared to Mixed-IT and Hyper-IT groups, the Iso-IT group had the best lytic effect (90.5 ± 1.6% vs. 78.4 ± 2.6% vs. 46.5 ± 3.3%, p < 0.001), lowest bleeding ratio (0.0 vs. 11.8 vs. 13.3, p < 0.001), and the lowest incidence rate of postthrombotic syndrome on 24 months (3.6 vs. 18.4 vs. 63.4%, p < 0.001) following CDT. The Iso-IT group had a significantly lower ratio of Fe3+ to total iron (93.1 ± 3.2% vs. 97.2 ± 2.1%, p = 0.034) and a higher content of fibrin (12.5 ± 5.3% vs. 4.76 ± 3.18%, p = 0.023) than Hyper-IT. Thrombus signal characteristics on T1-weighted black-blood MR is associated with CDT outcomes and possesses potential to serve as a noninvasive approach to guide treatment decision making in acute DVT patients. · Thrombus signal on T1-weighted black-blood MR is associated with lytic therapeutic outcome in acute DVT patients.. · Presence of iso-intense thrombus revealed by T1-weighted black-blood MRI is associated with successful thrombolysis, low bleeding ratio, and low incidence of the postthrombotic syndrome.. · T1-weighted thrombus signal characteristics may serve as a noninvasive imaging marker to predict CDT treatment outcomes and therefore guide treatment decision making in acute DVT patients..

Assessment of histological characteristics, imaging markers, and rt-PA susceptibility of ex vivo venous thrombi

Venous thromboembolism is a significant source of morbidity and mortality worldwide. Catheter-directed thrombolytics is the primary treatment used to relieve critical obstructions, though its efficacy varies based on the thrombus composition. Non-responsive portions of the specimen often remain in situ, which prohibits mechanistic investigation of lytic resistance or the development of diagnostic indicators for treatment outcomes. In this study, thrombus samples extracted from venous thromboembolism patients were analyzed ex vivo to determine their histological properties, susceptibility to lytic therapy, and imaging characteristics. A wide range of thrombus morphologies were observed, with a dependence on age and etymology of the specimen. Fibrinolytic inhibitors including PAI-1, alpha 2-antiplasmin, and TAFI were present in samples, which may contribute to the response venous thrombi to catheter-directed thrombolytics. Finally, a weak but significant correlation was observed between the response of the sample to lytic drug and its magnetic microstructure assessed with a quantitative MRI sequence. These findings highlight the myriad of changes in venous thrombi that may promote lytic resistance, and imaging metrics that correlate with treatment outcomes.

Iatrogenic Foreign Materials Associated with Retrieved Clot Tissue via Mechanical Thrombectomy.

Hydrophilic polymers and polytetrafluoroethylene liners, commonly used in the construction of endovascular devices, occasionally separate from devices with subsequent embolization. We determined the frequency of such materials in thrombus specimens retrieved by mechanical thrombectomy in patients with stroke. We retrospectively reviewed H&E-stained thrombus sections for presence and types of foreign materials. We identified 4 types of foreign materials-Type I: material was light green with refraction and had a homogeneous texture; type II: material was light gray and/or dark gray, thin, and loose or attenuated in texture; type III: material was light green with refraction, solitary in texture, irregular in shape, and was often associated with round or oval bubblelike particles and/or diffuse black particles; and type IV: material had homogeneous texture and was light pink or red. In addition, polymer materials from different layers of used mechanical thrombectomy catheters were compared with the foreign materials found in thrombus specimens. A total of 101 thrombi were evaluated. Foreign materials were found in 53 (52.5%) thrombus samples. The most common type was type I (92%), followed by type II (30%). The histopathologic features of the polymer materials from mechanical thrombectomy catheters were similar to the foreign materials found in thrombus specimens. The inner polytetrafluoroethylene liner and coating layer of catheters resembled type I and type II of the foreign materials, respectively. Foreign polymer materials are present in approximately half of retrieved thrombi, most commonly polytetrafluoroethylene from catheter liners and less from hydrophilic coatings.

Metagenomics Analysis of Thrombus Samples Retrieved from Mechanical Thrombectomy.

PurposeThe purpose of this study was to assess the microbiota in middle cerebral artery thrombi retrieved in mechanical thrombectomy arising out of symptomatic carotid plaque within 6 hours of acute ischemic stroke. Thrombi were subjected to next-generation sequencing for a bacterial signature to determine their role in atherosclerosis.Materials and MethodsWe included 4 human middle cerebral artery thrombus samples (all patients were male). The median age for the patients was 51±13.6 years. Patients enrolled in the study from Pacific Medical University and Hospital underwent mechanical thrombectomy in the stroke window period. All patients underwent brain magnetic resonance angiography (MRA) and circle of Willis and neck vessel MRA along with the standard stroke workup to establish stroke etiology. Only patients with symptomatic carotid stenosis and tandem lesions with ipsilateral middle cerebral artery occlusion were included in the study. Thrombus samples were collected, stored at –80 degrees, and subjected to metagenomics analysis.ResultsOf the 4 patients undergoing thrombectomy for diagnosis with ischemic stroke, all thrombi recovered for bacterial DNA in qPCR were positive. More than 27 bacteria were present in the 4 thrombus samples. The majority of bacteria were Lactobacillus, Stenotrophomonas, Pseudomonas, Staphylococcus, and Finegoldia.ConclusionGenesis of symptomatic atherosclerotic carotid plaque leading to thromboembolism could be either due to direct mechanisms like acidification and local inflammation of plaque milieu with lactobacillus, biofilm dispersion leading to inflammation like with pseudomonas fluorescence, or enterococci or indirect mechanisms like Toll 2 like signaling by gut microbiota.

Microbiota thrombus colonization may influence athero-thrombosis in hyperglycemic patients with ST segment elevation myocardialinfarction (STEMI). Marianella study

ObjectivesWe examined the association of the coronary thrombus microbiota and relative metabolites with major adverse cardiovascular events (MACE) in hyperglycemic patients with ST segment elevation myocardial infarction (STEMI). BackgroundHyperglycemia during STEMI may affect both development and progression of coronary thrombus via gut and thrombus microbiota modifications. MethodsWe undertook an observational cohort study of 146 first STEMI patients treated with primary percutaneous coronary intervention (PPCI) and thrombus-aspiration (TA). Patients were clustered, based on admission blood glucose levels, in hyperglycemic (≥140 mg/dl) and normoglycemic (<140 mg/dl). We analyzed gut and thrombus microbiota in all patients. Moreover, we assessed TMAO, CD40L and von Willebrand Factor (vWF) in coronary thrombi. Cox regressions were used for the association between Prevotellaspp. and TMAO terziles and MACE. MACE endpoint at 1 year included death, re-infarction, unstable angina. ResultsIn fecal and thrombus samples, we observed a significantly different prevalence of both Prevotellaspp. and Alistipesspp. between patients with hyperglycemia (n = 56) and those with normal glucose levels (n = 90). The abundance of Prevotella increased in hyperglycemic vs normoglycemic patients whereas the contrary was observed for Alistipes. Interestingly, in coronary thrombus, the content of Prevotella was associated with admission blood glucose levels (p < 0.01), thrombus dimensions (p < 0.01), TMAO, CDL40 (p < 0.01) and vWF (p < 0.01) coronary thrombus contents. Multivariate Cox-analysis disclosed a reduced survival in patients with high levels of Prevotella and TMAO in coronary thrombus as compared to patients with low levels of Prevotella and TMAO, after 1-year follow up. ConclusionsHyperglycemia during STEMI may increase coronary thrombus burden via gut and thrombus microbiota dysbiosis characterized by an increase of Prevotella and TMAO content in thrombi.Clinical Trial Registration: NCT03439592. September 30, 2016.Ethic Committee Vanvitelli University: 268/2016.

Intrathrombotic appearances of AQP-1 and AQP-3 in relation to thrombus age in murine deep vein thrombosis model.

Aquaporins (AQPs) are membrane-bound proteins for water transportation and are useful for diagnosing drowning and wound vitality in forensic pathology. Here, we examined intrathrombotic expression of AQP-1 and AQP-3 using deep vein thrombosis models in mice. To perform immunohistochemical analyses, we used anti-AQP-1 and anti-AQP-3 antibodies. In thrombus samples with the post-ligation intervals of 1 to 5 days, AQP-1+ areas were over 70%. At 7 days after the IVC ligation, AQP-1+ areas became less than 50%, eventually decreasing to 11% at 21 days. At 3 days after the IVC ligation, AQP-3+ cells started to appear from the peripheral area. Thereafter, the positive cell number progressively increased and reached to a peak at 10 days after the IVC ligation. When the intrathrombotic AQP-1+ area was as large as the intrathrombotic collagen area or smaller, it would indicate a thrombus age of ≥ 10 days. AQP-3+ cell number of > 30 would indicate a thrombus age of 10–14 days. Collectively, our study implied that the detection of AQP-1 and AQP-3 would be useful for the determination of thrombus age.

The expression of myeloperoxidase in thrombi is associated with reduced heme oxygenase-1 induction and worse left ventricular remodeling in patients with acute ST-elevation myocardial infarction.

BackgroundMyeloperoxidase (MPO) secreted by neutrophils is the enzyme that kills bacteria and other pathogens. Acute myocardial infarction (AMI) is usually caused by thrombosis in response to vulnerable plaque rupture. Circulating MPO was found to be associated with increased mortality in AMI patients. However, the relationship between MPO in thrombi and the prognosis of AMI patients remains unknown.HypothesisMPO expression in thrombi is associated with the prognosis of patients who underwent primary percutaneous coronary intervention (PCI) after AMI.MethodsThis study included 41 consecutive patients with acute ST‐elevation myocardial infarction, who successfully underwent primary PCI, during which we collected thrombi remaining in the culprit artery using aspiration catheters. These thrombus samples were fixed, and immunohistochemical staining against MPO and heme oxygenase‐1 (HO‐1) was conducted. Enrolled patients were divided into two groups based on the induction of thrombotic MPO, which was quantified using Image J software.MethodsWe observed that increased MPO was associated with lower left ventricular ejection fraction (LVEF) and worse LV remodeling in AMI patients. Instead, patients with decreased thrombotic MPO induction had a considerable improvement in LVEF 1 month after discharge (54.4 ± 2.0% vs. 61.1 ± 2.3%, p < 0.01). In the MPO group, a reduction in the thrombotic HO‐1 level contributed to the development of adverse LV remodeling. Logistic regression showed that MPO was a considerable risk factor for adverse LV remodeling (adjusted OR 3.70, p < 0.05).ConclusionMPO expression in thrombi is associated with reduced LVEF and deteriorated LV remodeling in AMI patients, which may be due to HO‐1 suppression in thrombi.

The application of autophagy to thrombus age estimation in murine deep vein thrombosis model.

We immunohistochemically examined the intrathrombotic dynamics of autophagy during thrombogenesis using murine deep vein thrombosis (DVT) models. To perform the immunohistochemical analyses, we used anti-LC3 antibody and anti-p62 antibody for detecting the intrathrombotic autophagic functions. We estimated dynamics of the intrathrombotic autophagy as LC3+ cell number (×1000, five fields) with the thrombus ages (each group n = 5). The number of LC3+ cells was once decreased at 3 days, and then increased until 10 days. On the contrary, the number of p62+ cells progressively increased until 10 days after the inferior vena cava (IVC) ligation, and then gradually decreased. Especially, in all of thrombus samples with the postligation intervals of 5-10 days, both numbers were larger than 10. Subsequently, we compared the number of LC3+ cells to that of p62+ cells. Although, at 1 day after the IVC ligation, LC3+ cell number significantly exceeded p62+ cell number, the former was significantly or relatively less than the latter at 3 days or more after the IVC ligation. Thus, positive cells of > 10 in both LC3 and p62 indicated the thrombus age of 5-10 days. Upon comparison of immunopositive cells in LC3 and p62, the p62/LC3 ratio was > 1.0 in 29 out of 30 thrombus samples aged 3-21 days, and all of 1-day-old thrombus had the p62/LC3 ratio of < 0.5. Thus, the ratio of > 1.0 and that of < 0.5 could indicate thrombus age of 3 days or more and that of 1 day, respectively. Collectively, our study implied that the detection of autophagy-related molecules such as LC3 and p62 would be useful for the determination of thrombus age.

Immunogenic Heterogeneity of Renal Cell Carcinoma With Venous Tumor Thrombus

ObjectiveTo perform immune-cell enumeration and programmed death-ligand 1 (PD-L1) expression in clear cell renal cell carcinoma (cc-RCC) with tumor thrombus (TT) to guide therapeutic decisions. MethodsAfter obtaining IRB approval and surgical consent, 6 patients underwent radical nephrectomy with venous tumor thrombectomy. We utilized RNA Sequencing to obtain RNAseq expression profiles. Computational calculation and enumeration of immune cells were performed using CIBERSORT, xCell, and ingenuity pathway analysis software. Statistical assessment was conducted using a t test, chi-square, ANOVA and Spearman rank correlations using SPSS v21. ResultsWe observed a higher proportion of M1 macrophages in the primary tumor and tumor thrombus, while we noted no difference in M2 macrophages despite M2 representing a high number in thrombus samples. (ANOVA, P = .032, and P = .89, respectively). Validation with xCell and ingenuity pathway analysis analysis showed a high involvement of macrophages. We observed a higher number of M1 macrophages (CIBERSORT mean 0.11 vs 0.03, P < 0.01) and (nonactivated) resting Natural Killer (NK) cells (0.077 vs 0.017, P = .02) associated PD-L1 high expression of the primary tumor. PDL1 expression was variable without differences in tumor stage, level, or immune cell detection. We observed an inverse correlation of mean platelet volume with PD-L1 expression within the primary tumor (Spearman, −0.89, P = 02) and the TT (Spearman, −0.77, P = 0.07). ConclusionRenal tumor thrombus has higher levels of M1 macrophages that could be utilized as additional targets for future drug development. The PD-L1 expression on clear cell RCC biopsy may not represent its corresponding TT. Future studies are needed to confirm mean platelet volume as a potential blood-based biomarker for PD-L1 expression in RCC.

Abdominal Aortic Aneurysms Growth Is Associated With High Concentrations of Plasma Proteins in the Intraluminal Thrombus and Diseased Arterial Tissue.

Objective- Porosity of the intraluminal thrombus (ILT) is believed to convey biologically active components from the bloodstream toward the aneurismal wall. Accumulation of molecules in the abdominal aortic aneurysmatic tissue may influence vascular protein turnover and regulate abdominal aortic aneurysm growth. We sought to identify proteins with concentrations in the ILT and the abdominal aortic aneurysm wall which associate with aneurysmal expansion rate. Approach and Results- Proteomic analysis by liquid chromatography tandem-mass spectrometry of separated wall and ILT samples was correlated with preoperative aneurysmal growth rate in 24 individuals operated electively for infrarenal abdominal aortic aneurysm. The median preoperative growth rate was 3.8 mm/y (interquartile range, 3) and the mean observational time was 3.3±1.7 years. Plasma components dominated the group of proteins with tissue concentrations, which correlate positively with growth rates ( P<0.001, Fisher exact test, both in the ILT and the wall). In contrast, in the wall and thrombus samples, ECM (extracellular matrix) proteins were significantly more prevalent in the group of proteins with negative correlations to growth rates ( P<0.05, Fisher exact test). Similarly, a long series of proteins, related to cellular functions correlated negatively to growth rates. Conclusions- When the preoperative aneurysmatic growth rate has been high, the concentration of many plasma proteins residing in the ILT and the aneurysmatic tissue is also high, compatible with the hypothesis of increased tissue porosity and accumulation of plasma components as a driver of aneurysm expansion. Moreover, many matrix and cellular proteins which are found in high concentrations in slower-growing aneurysms provides new knowledge about potential treatment targets.