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Platelet activation and inhibition in atherothrombosis and inflammation
Detailing the role of neutrophils in atherothrombosis
TP53-MUTANT CLONAL HEMATOPOIESIS ACCELERATES EXPERIMENTAL ATHEROSCLEROSIS DEVELOPMENT.
Abstract
Background and Aims
Acquired mutations in hematopoietic cells that lead to clonal hematopoiesis (CH) are emerging as new drivers of atherosclerotic cardiovascular disease. The TP53 gene, which encodes the tumor suppressor protein p53, is one of the most frequently mutated genes in individuals exhibiting CH. Here, we investigated the effects of CH driven by inactivating mutations in p53 on experimental atherosclerosis development.
Methods
Chimeric Ldlr-/- mice carrying 20% Trp53-/- hematopoietic cells (20% KO BMT mice) were generated through competitive bone marrow (BM) transplants. White blood cells (WBC) and BM progenitors were analyzed by flow cytometry. Atherosclerotic plaque development and composition were evaluated by histological techniques. Cultured murine BM derived macrophages (BMDM) were used to perform mechanistic studies.
Results
Trp53-/- cells expanded progressively in WBCs and BM progenitors, which led to 39% larger atherosclerotic plaques in the aortic root (p=0.009). Increased plaque macrophage content was observed in 20% KO BMT mice, in parallel with higher proliferation of Trp53-/- macrophages within the arterial wall, as revealed by Ki67 immunostaining. Bulk RNA sequencing and in vitro analysis of MCSF-stimulated BMDM showed that p53 deficiency leads to widespread changes in the expression of cell proliferation-related genes and results in accelerated cell cycle progression. Additionally, Trp53-/- BMDM exhibit a marked downregulation of cell-death-related transcripts and reduced apoptosis after UV radiation or MCSF deprivation. Consistent with these findings, 20% KO BMT mice displayed less apoptotic nuclei in atherosclerotic plaques.
Conclusions
TP53 mutation-driven CH accelerates experimental atherosclerosis development and increases plaque macrophage burden by modulating macrophage proliferation and apoptosis.
IN VIVO DETECTION OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (UPAR) EXPRESSION IN ARTERIAL ATHEROGENESIS USING [64CU]CU-DOTA-AE105 POSITRON EMISSION TOMOGRAPHY
Abstract
Background and Aims
Molecular imaging provides an accurate choice to image development of atherosclerosis. The aim of the study was to non-invasively visualize urokinase-type plasminogen activator receptor (uPAR) expression in atherosclerosis by a novel uPAR-targeting PET tracer, [64Cu]Cu-DOTA-AE105.
Methods
We used molecular biology techniques to quantify uPAR expression in an in vitro assay for THP-1 derived cells and in freshly excised plaques. Furthermore, we visualized uPAR expression on PET/CT scans of patients enrolled in a Phase 1 trial divided into groups based on coronary artery calcium scores (CAC).
Results
Cells in an in vitro assay displayed a significantly upregulated uPAR expression upon stimulation with 7-ketocholesterol and IFN- ɣ (5.2-fold upregulation, p<0.0001 by a one-way ANOVA & Tukey’s test, Figure A) by flowcytometric analysis. Human atherosclerotic plaques underwent flowcytometric and microarray analyses which showed 73.9 ± 2.9% cells expressing uPAR and had a greater than 7-fold higher gene expression of Plasminogen Activator Urokinase Receptor (PLAUR, p=0.002), Integrin Subunit Alpha X (ITGAX, p=0.0008), and Cluster of Differentiation 163 (CD163, p<0.0001, Figure B). Lastly, in a retrospective analysis of patients, the tissue-to-background ratios (TBRmax) in arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4±0.1 vs 1.7±0.1, p=0.057), significantly higher in the ascending aorta (2.7±0.1 vs 2.0±0.1, p=0.038) and the abdominal aorta (3.2±0.2 vs 2.0±0.2, p=0.038, Figure D&E) by a Mann-Whitney test.
Conclusions
uPAR is abundantly expressed by cells in atherosclerotic plaques and can be visualized by the novel PET tracer [64Cu]Cu-DOTA-AE105 that may non-invasively image atherogenesis.
LIPOPROTEIN(A) AND SARS-COV-2 INFECTIONS: RISK FOR ISCHEMIC HEART DISEASE AND THROMBOEMBOLIC EVENTS.
Abstract
Background and Aims
SARS-CoV-2 positive patients show high frequency of thromboembolic complications. Comorbidities like ischemic heart disease (IHD) associate with worse outcome. Since lipoprotein(a) [Lp(a)] is a strong risk factor for IHD and has possibly thrombogenic properties, we investigated in UK Biobank whether SARS-CoV-2 infections alter the Lp(a)‑driven risk for IHD and thromboembolic events.
Methods
Follow-up study from March 16, 2020 to November 30, 2020 in 6,937 positive patients and 435,104 population controls (negative or non‑tested) from UK Biobank. We compared the median of baseline Lp(a) (measured long before the pandemic) in the two groups by Wilcoxon test and the frequency of IHD and thromboembolic events by chi‑square test. The Lp(a)‑driven risks for IHD and thromboembolic events were estimated by logistic regression models using Lp(a) both as a continuous variable and in categories.
Results
The Lp(a) concentration were similarly distributed in positive patients and population controls. Thromboembolic events were five‑times more frequent in SARS-CoV-2 positive patients (1.54% vs. 0.31%, p=4.68e-74), but did not associate with Lp(a) (OR=1.00; p=0.90). However, the IHD risk was significantly associated with the Lp(a) concentration in both groups. Interestingly, the risk increase was steeper in SARS‑CoV‑2 positive patients (interaction p-value=0.030) (Figure). The OR for IHD of the top 5% to the bottom 20% of the Lp(a) distribution was 48% higher within positive patients (OR[95%CI]=2.22[1.40‑3.54], p=0.00067) versus population controls (OR[95%CI]=1.50[1.34‑1.69), p=2.20e-12).
Conclusions
SARS-CoV-2 infections enhance the association between Lp(a) and IHD. Baseline Lp(a) does not affect the risk for thromboembolic events in SARS-CoV-2 patients.