Fibrin clot characteristics and anticoagulant response in a SARS-CoV-2-infected endothelial model.

Coronavirus disease 2019 (COVID-19) patients have increased thrombosis risk. With increasing age, there is an increase in COVID-19 severity. Additionally, adults with a history of vasculopathy have the highest thrombotic risk in COVID-19. The mechanisms of these clinical differences in risk remain unclear. Human umbilical vein endothelial cells (HUVECs) were infected with SARS-CoV-2, influenza A/Singapore/6/86 (H1N1) or mock-infected prior to incubation with plasma from healthy children, healthy adults or vasculopathic adults. Fibrin on surface of cells was observed using scanning electron microscopy, and fibrin characteristics were quantified. This experiment was repeated in the presence of bivalirudin, defibrotide, low-molecular-weight-heparin (LMWH) and unfractionated heparin (UFH). Fibrin formed on SARS-CoV-2 infected HUVECs was densely packed and contained more fibrin compared to mock-infected cells. Fibrin generated from child plasma was the thicker than fibrin generated in vasculopathic adult plasma (p = 0.0165). Clot formation was inhibited by LMWH (0.5 U/ml) and UFH (0.1-0.7 U/ml). We show that in the context of the SARS-CoV-2 infection on an endothelial culture, plasma from vasculopathic adults produces fibrin clots with thinner fibrin, indicating that the plasma coagulation system may play a role in determining the thrombotic outcome of SARS-CoV-2 infection. Heparinoid anticoagulants were most effective at preventing clot formation.

Acute Routine Leukocyte and Neutrophil Counts Are Predictive of Poststroke Recovery at 3 and 12 Months Poststroke: An Exploratory Study.

Background and Aims. White blood cell (WBC) and neutrophil counts (NC) are common markers of inflammation and neurological stroke damage and could be expected to predict poststroke outcomes. Objective. The aim of this study was to explore the prognostic value of early poststroke WBC and NC to predict cognition, mood, and disability outcomes at 3 and 12 months poststroke. Methods. Routine clinical analyses WBC and NC were collected at 3 time points in the first 4 days of hospitalization from 156 acute stroke patients. Correlations using hierarchical or ordinal regressions were explored between acute WBC and NC and functional recovery, depression, and cognition at 3 and 12 months poststroke, after covarying for age and baseline stroke severity. Results. We found significant increases in NC between <12 hours and 24 to 48 hours time points (P = .05). Hierarchical regressions, covaried for age and baseline stroke severity, found that 24 to 48 hours WBC (P = .05) and NC (P = .04) significantly predicted 3-month cognition scores. Similarly, 24 to 48 hours WBC (P = .05) and NC (P = .02) predicted cognition scores at 12 months. Increases in WBC and NC were predictive of increased cognition scores at both 3 and 12 months (positive recovery) though there were no significant associations between WBC and NC and disability or depression scores. Conclusions. Routine acute stroke clinical laboratory tests such as WBC and NC taken between 24 and 48 hours poststroke are predictive of cognition poststroke. It is interpreted that higher rapid immunological activation in the acute phase is an indicator for the trajectory of positive stroke recovery.

Longitudinal Stroke Recovery Associated With Dysregulation of Complement System-A Proteomics Pathway Analysis.

Currently the longitudinal proteomic profile of post-ischemic stroke recovery is relatively unknown with few well-accepted biomarkers or understanding of the biological systems that underpin recovery. We aimed to characterize plasma derived biological pathways associated with recovery during the first year post event using a discovery proteomics workflow coupled with a topological pathway systems biology approach. Blood samples (n = 180, ethylenediaminetetraacetic acid plasma) were collected from a subgroup of 60 first episode stroke survivors from the Australian START study at 3 timepoints: 3-7 days (T1), 3-months (T2) and 12-months (T3) post-stroke. Samples were analyzed by liquid chromatography mass spectrometry using label-free quantification (data available at ProteomeXchange with identifier PXD015006). Differential expression analysis revealed that 29 proteins between T1 and T2, and 33 proteins between T1 and T3 were significantly different, with 18 proteins commonly differentially expressed across the two time periods. Pathway analysis was conducted using Gene Graph Enrichment Analysis on both the Kyoto Encyclopedia of Genes and Genomes and Reactome databases. Pathway analysis revealed that the significantly differentiated proteins between T1 and T2 were consistently found to belong to the complement pathway. Further correlational analyses utilized to examine the changes in regulatory effects of proteins over time identified significant inhibitory regulation of clusterin on complement component 9. Longitudinal post-stroke blood proteomics profiles suggest that the alternative pathway of complement activation remains in a state of higher activation from 3-7 days to 3 months post-stroke, while simultaneously being regulated by clusterin and vitronectin. These findings also suggest that post-stroke induced sterile inflammation and immunosuppression could inhibit recovery within the 3-month window post-stroke.

A Pathway Proteomic Profile of Ischemic Stroke Survivors Reveals Innate Immune Dysfunction in Association with Mild Symptoms of Depression - A Pilot Study.

Depression after stroke is a common occurrence, raising questions as to whether depression could be a long-term biological and immunological sequela of stroke. Early explanations for post-stroke depression (PSD) focused on the neuropsychological/psychosocial effects of stroke on mobility and quality of life. However, recent investigations have revealed imbalances of inflammatory cytokine levels in association with PSD, though to date, there is only one published proteomic pathway analysis testing this hypothesis. Thus, we examined the serum proteome of stroke patients (n = 44, mean age = 63.62 years) and correlated these with the Montgomery-Åsberg Depression Rating Scale (MADRS) scores at 3 months post-stroke. Overall, the patients presented with mild depression symptoms on the MADRS, M = 6.40 (SD = 7.42). A discovery approach utilizing label-free relative quantification was employed utilizing an LC-ESI-MS/MS coupled to a LTQ-Orbitrap Elite (Thermo-Scientific). Identified peptides were analyzed using the gene set enrichment approach on several different genomic databases that all indicated significant downregulation of the complement and coagulation systems with increasing MADRS scores. Complement and coagulation systems are traditionally thought to play a key role in the innate immune system and are established precursors to the adaptive immune system through pro-inflammatory cytokine signaling. Both systems are known to be globally affected after ischemic or hemorrhagic stroke. Thus, our results suggest that lowered complement expression in the periphery in conjunction with depressive symptoms post-stroke may be a biomarker for incomplete recovery of brain metabolic needs, homeostasis, and inflammation following ischemic stroke damage. Further proteomic investigations are now required to construct the temporal profile, leading from acute lesion damage to manifestation of depressive symptoms. Overall, the findings provide support for the involvement of inflammatory and immune mechanisms in PSD symptoms and further demonstrate the value and feasibility of the proteomic approach in stroke research.

Inflammasome activity is essential for one kidney/deoxycorticosterone acetate/salt-induced hypertension in mice.

BACKGROUND AND PURPOSE: Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1ß and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1ß and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH: Wild-type and inflammasome-deficient ASC(-/-) mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. KEY RESULTS: 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1ß, as well as protein levels of active caspase-1 and mature IL-1ß. Following treatment with 1K/DOCA/salt, ASC(-/-) mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. CONCLUSIONS AND IMPLICATIONS: Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1ß pathway as a potential therapeutic target in hypertension.