tbuckley/cabot-search huggingface dataset:
Circulating LIGHT/TNFSF14 levels are negatively associated with cardiovascular mortality, while intra-plaque levels are associated with symptomatic cerebrovascular disease
Authors: A Shami, A Edsfeldt, J Nilsson, I Goncalves
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): The Swedish Heart and Lung Foundation, Åke Wiberg's Foundation Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it is associated with a high-risk plaque phenotype and cardiovascular events is not known. Purpose This study investigates the association between intra-plaque and circulating LIGHT levels and cardiovascular disease. Method Plaque levels of LIGHT/TNFSF14 were measured in plasma and homogenised carotid endarterectomy plaques by Proximity Extension Assay in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=557 and 201, respectively). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through immunohistochemistry. Multivariate Cox proportional hazards analysis was performed on normalized (logarithmically transformed) values. Results Plaque, but not circulating, levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, intra-plaque levels of LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, fibromodulin, ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7) and matrix metalloproteinases 1, 2, 9 and 10. While plaque LIGHT levels were not predictive of cardiovascular death, circulating LIGHT levels were negatively associated with risk of cardiovascular death (after 6 ½ years) after correcting for the possible confounders age, sex, cerebrovascular symptoms, diabetes, high-sensitivity C-reactive protein, inflammatory disease, hypertension and smoking (hazard ratio=0.032, confidence interval=0.420-0.951, p=0.028). Conclusion Though intra-plaque LIGHT levels are associated with a rupture-prone plaque phenotype and pre-operative symptoms, circulating levels are associated with a lower risk of future cardiovascular mortality. This suggest that upregulation of LIGHT in atherosclerotic plaques may occur as part of a reparative rupture response and, systemically, reflect efficient wound-healing capacity.
Human atherosclerotic plaque macrophages associate with clinical presentation and follow diverse lineage differentiation routes
Authors: K Prange, G Bel-Bordes, M A C Depuydt, R W M Kempkes, M D M De Jong, B M Mol, B Slutter, I Bot, A E Neele, D P V De Kleijn, G J De Borst, J Kuiper, G Pasterkamp, M Mokry, M P J De Winther
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Research Council Veni grant. Netherlands Heart Foundation Atherosclerosis is characterized by inflammation and lipid accumulation, leading to complications such as stroke and myocardial infarction. Various macrophage subsets are present in human atherosclerotic plaques. However, it is unclear how subsets are interrelated and contribute to the disease process and its clinical outcomes. Here, we employed single-cell RNA-seq (scRNA-seq) on blood and plaque material from carotid endarterectomy patients enrolled in the AtheroExpress (AE) cohort to define regulatory pathways of macrophage recruitment, differentiation, and development. Additionally, we used these scRNA-seq data to deconvolute a large AE bulk RNA-seq cohort to allow correlating cellular subsets in plaques with clinical traits. Interestingly, we found that macrophages and their subsets are the only cell population significantly associated with major adverse cardiac events (MACE) during a 3-year follow-up period. Subsequently, we could divide plaque macrophages into 3 populations: inflammatory, tissue-resident / lipid-associated (TREM2 high), and foamy macrophages (TREM1 high). While the latter two are correlated with MACE, the inflammatory macrophages are inversely correlated with statin use. Cellular fate analyses revealed two major paths of macrophage development in the plaque. First, non-classical monocytes entering the plaque, turning into inflammatory cells before becoming foamy and eventually dying. Second, resident-like macrophages that turn into either inflammatory- or lipid-associated macrophages, before meeting their foamy demise. Together, these findings provide important insights on macrophages in atherosclerosis and suggest new leads to discover therapeutic targets for this disease.
Proteomic profiling reveals higher presence of glycolytic enzymes in vulnerable atherosclerotic lesions
Authors: K C A Palm, K Theofilatos, S Van Der Laan, E Diez Benavente, G J De Borst, D P V De Kleijn, J Wotja, M Mayr, H Den Ruijter, M Mokry, G Pasterkamp
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq PlaqOmics Background Our definition of vulnerable plaques is predominantly driven by histological analysis. The molecular mechanisms behind the vulnerable histological characteristics are not fully understood. Here we use mass spectrometry-based proteomics data, to identify molecular mechanisms linked to vulnerable plaques. Methods Atherosclerotic lesions from two hundred patients undergoing endarterectomy surgery were collected (149 male and 51 female plaques). From a total of two hundred plaque samples, extracellular matrix proteins were extracted and processed for untargeted proteomics analysis. The resulting protein levels were linked to existing histological characteristics, transcriptomic, and clinical data. Results We detected 1499 proteins in atherosclerotic lesions, which were mostly affiliated with biological processes including extracellular matrix organization, coagulation, and peptidase activity. Linear regression identified 240 proteins associated with plaque vulnerability index (a semi-quantitative score summarizing the presence of SMC cells, macrophages, plaque hemorrhage, and fat in the plaque). Among the proteins associated with high plaque vulnerability index we identified an overrepresentation of enzymes involved in glycolysis – including the rate limiting enzymes HK3 (beta =0.40 p =0.003) and PKM (beta =0.19 p =0.009). This was driven by macrophage numbers and fat content in the plaque. Furthermore, data from a validation cohort of 120 patients from the Medical University of Vienna, Austria, revealed that HK3 and PKM were associated with plaque progression (p =&amp;lt;0.001, p =0.017) and clinical symptoms (p = &amp;lt;0.001, p = &amp;lt;0.001). Conclusion Enzymes involved in the glycolysis process show higher presence in vulnerable plaques. This suggests an important role for energy metabolism in plaque vulnerability.
Role of inflammatory populations and their intermediates in post-ischemic adverse remodeling in rat model of myocardial infarction
Authors: E Bevilacqua, D Falcon, A Ordonez-Fernandez, R Del Toro, T Smani
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía- Fondos FEDER Background Compelling evidence demonstrated the critical role of inflammation in ventricular adverse remodeling after acute myocardial infarction. However, it remains unclear how inflammatory cell population and their intermediates participate in the progress of the disease. Purpose This study aims to elucidate the role of inflammatory population in cardiac remodeling after myocardial infarction in a rat model, focusing on their role in fibrosis. Methods We used a surgical procedure by transient ligation of the left descendant coronary artery, to evoke ischemia-reperfusion (I/R) in rats. We performed FACS analysis to evaluate counts of neutrophils and two monocytes’ subsets: CD43lowHis48high (classical monocytes), and CD43highHis48low (non-classical monocytes), which are the most pro-inflammatory subset in peripheral blood. Subsequently, we performed immunofluorescence to detect pro-inflammatory M1 and M2 macrophages (CD68+ and CD163+) in the rat infarcted myocardium. Moreover, we sorted the two monocyte subsets and performed genes and microRNAs Array. Results FACS analysis of neutrophils and monocytes subsets showed an increased level of these cells in the rat model of myocardial infarction, one week after the post-ischemic event. In parallel, the infiltration of M1 macrophages was increased in the myocardium until 1 week after surgery, indicating a long extravasation of inflammatory cells after the ischemic event. Moreover, co-culture of fibroblasts and cardiomyocytes with intermediates released by inflammatory cells subjected to I/R increased the expression of fibrotic and apoptotic genes. In addition, the analysis of genes’ Array by GSEA indicated that CD43highHis48low monocytes had an enrichment score in pathways associated with angiogenesis, fibrosis and inflammation, while CD43lowHis48high population exhibited an enrichment in cytokines and inflammation, apoptosis and fibrosis. Finally, we found significant overexpression of microRNAs, for instance, miR-27a-3p, miR-29b-3p, miR-30e-5p and miR-194-5p in CD43highHis48low 24h after the I/R procedure. Those miRNAs are predicted to modulate pro-fibrotic genes. Conclusions The level of inflammatory cell populations is increased in I/R rat model. Moreover, these cells infiltrate in the myocardium likely to change the transcriptome of resident cells towards a pro-apoptotic and pro-fibrotic phenotype.
Caveolin-dependent endocytosis regulates arterial calcification
Authors: A Gorgels, J Simon, N Hense, N Marx, C Goettsch
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): SFB219 Introduction Patients with chronic kidney disease (CKD) have a significantly elevated cardiovascular risk, which is primarily due to premature vascular aging with arterial calcification - a substantial public health burden. Calcifying extracellular vesicles (EVs) are required for the onset and progression of calcification. Endocytosis and exocytosis are important for EV genesis and cargo loading, thus controlling EV release and propensities. Purpose We aim to investigate endocytosis, endosomal ripening, and exocytosis as critical mechanisms of EV-dependent mineralization. Methods and Results First, we characterized EVs from calcifying vascular smooth muscle cells (SMCs) compared to control SMCs. Western blot analysis revealed an increase in tissue nonspecific alkaline phosphatase (TNAP) among calcifying EVs but not in flotillin abundance. When compared to control EVs, calcifying EVs showed higher TNAP activity. Flow cytometry and a turbidity assay were then used to evaluate EV mineral cargo and aggregation potential. Compared to the control, EVs produced from SMCs cultivated under calcifying conditions had 5-fold more mineral-positive EVs (p=0.004) and a 3-fold higher nucleation potential (p&amp;lt;0.001). Next, endocytosis was blocked by targeting Dynamin-1 and –2, which inhibited caveolin- and clathrin-dependent endocytosis (Dynasore hydrate), or targeting clathrin-terminal domain, which inhibited solely clathrin-dependent endocytosis (Pitstop 2). Dynasore reduced matrix mineralization dose-dependently and lowered mineral-positive EVs by 18%, whereas Pitstop 2 had no effect, suggesting a role of caveolin-dependent endocytosis in EV-mediated calcification. Indeed, Cav-1 silencing inhibited matrix mineralization and Cav-1 protein was more abundant in calcifying EVs than in control EVs. Dynasore and Pitstop-2 did not affect the EV number measured by nanoparticle tracking analysis. Inhibition of endosomal ripening by Rab7 inhibition did not affect SMC calcification. Exocytosis was inhibited by Rab27a inhibition, which reduced mineral deposition (-32%, p=0.017) without affecting EV mineral or EV quantity. Conclusion EVs released from calcifying SMCs showed a higher abundance of mineral and TNAP proteins, suggesting a role in mineral transport and deposition. Our data suggest that caveolin-dependent endocytosis is essential in EV-mediated SMC calcification. Mineral cargo in EVs is independent of Rab7 and Rab27a pathways.
Role of miRNAs regulation of Orai1 and store operated Ca2+ entry in vascular restenosis
Authors: M Martin Bornez, B Dominguez Liste, D Falcon Boyano, T Smani
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agencia Estatal de Investigación Background The main cause of restenosis is attributed to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Orai1 is one of the main molecular components of Store-Operated Calcium Entry (SOCE), playing a key role in VSMCs proliferation during neointima formation and vascular remodeling. Recent studies have demonstrated the implication of microRNAs (miRNAs) in VSMCs proliferation and migration, however, the role of these miRNAs as regulators of SOCE during vascular remodeling has not been described. Purpose This study aims to determine whether miRNAs regulate the intracellular Ca2+ handling, focusing on SOCE, during neointima formation. Methods Experiments were conducted in an animal rat model following the "Principles of laboratory animal care" and according to the Spanish law. The neointima development was assessed after balloon injury of carotid arteries and confirmed by haematoxylin and eosin staining, and αSMA staining of tissue sections up to 3 weeks after surgery. miRNAs array and RT-qPCR was used to examine the expression of miRNAs. Dual-Luciferase reporter assay was performed in A7R5 VSMCs line. Results Injured arteries showed significant increase in the expression of Orai1, as compared to non-injured arteries, indicating their possible participation in the neointima layer formation. Microarray of miRNAs analysis of the injured carotids compared to control carotids showed significant increase in the expression of miRNAs related to signalling pathways involving cell proliferation and migration, cell differentiation, or VSMCs contraction. Of these, we confirmed the significant increase of miR-18a-5p, miR-20a-5p, miR-20b-5p and miR-17-5p, which belong to widely studied miR-17-92 cluster involved in different cell proliferation. Moreover, we found that mimics of those miRNAs were able to regulate differentially the activity of the Orai1 promotor in A7R5 cell line, indicating that Orai1 can be targeted by miRNAs during in VSMCs. Conclusions In conclusion, our data confirms that Orai1 is upregulated during neointima formation associated with miRNAs dysregulation. Moreover, we found that Orai1-dependent SOCE can be finely regulated by miRNAs during restenosis.
Characterization of senescence in human cardiac fibroblasts
Authors: A Costa, D Ceresa, P Altieri, P Malatesta, P Ameri
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements None. Background Recent studies suggest that senescence of cardiac fibroblasts (cFib) plays a significant role in cardiac aging and disease. However, senescence of cultured cFib in response to classical pro-senescent stimuli has not been characterized yet. Purpose We thoroughly studied oncogene- and stress-induced senescence, as well as replicative senescence, in primary human cFib. Methods Human cFib were isolated from specimens of right atrial appendages, collected during cardiac surgery with extracorporeal circulation. These cells were subjected to oncogene-induced senescence by overexpression of HRasV12, were exposed to ionizing radiation (IR, 5 Gy) to recapitulate stress-induced senescence, and were passaged until replicative senescence was reached (P12-P14). Senescence was assessed by staining for senescence associated (SA) β-galactosidase and EdU and by RT-PCR for CDKN1A, CDKN2A, and LamB1. Results The main results are summarized in the graphical abstract. HRasV12 overexpression, IR, and prolonged culturing consistently caused senescence of cFib: in fact, all senescent models showed a significant increase in SA-β-gal activity and CDKN1A expression and a decrease in EdU uptake and LamB1 expression over control cells. Interestingly, cFib with HRasV12 overexpression exhibited a peculiar morphological phenotype with vacuoles and showed higher levels of the typical markers of senescence compared to control and to IR-treated or extensively passaged cFib. Moreover, only cFib with HRasV12 overexpression showed a strong downregulation of CDKN2A. Conclusions Cultured primary human cFib undergo senescence in response to established inducers of senescence. HRasV12 overexpression uniquely causes vacuolization of senescent cFib, which is currently being further characterized by transmission electron microscopy.
Incomplete microvascular reperfusion in a swine model of recanalized acute ischemic stroke
Authors: M Wang, A Taha, J D Raben, R Noort, M Stam, D J Duncker, D W J Dippel, J Bobi, H M M Beusekom
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): CONTRAST CONSORTIUM (CVON, Dutch Heart Foundation) Background Incomplete microvascular reperfusion (IMR) could be a detrimental factor impairing regained functionality in stroke patients despite early recanalization. However, the presence of this phenomenon remains to be ascertained. Gyrencephalic large-animal models of acute ischemic stroke (AIS) such as swine are gaining attention as a next step in translation of preclinical to clinical stroke research, with brain anatomy and hemostasis more similar to humans. However, the occurrence and relevance of IMR in swine models of AIS is not well described yet, since most models do not include recanalization. Purpose We aimed to study the presence of IMR after brain ischemia in a translational swine model of AIS. Methods Anesthetized female Yorkshire-Landrace pigs (n=23) underwent craniotomy to occlude the right-sided middle cerebral arteries with aneurysm clips. Clips were released after 1 (n=5), 2 (n=5), and 4 (n=5) hours of occlusion to allow recanalization for 4 hours (recanalized AIS, n=15) or left in place for 8 hours (non-recanalized AIS, n=5). Three animals underwent the craniotomy without vessel clipping (Sham, n=3). 3D angiography was used to confirm occlusion and recanalization. Brains were collected after sacrifice, stained with TTC and then processed for histologic analysis. Infarct size was determined by TTC staining and expressed as percentage of the infarcted hemisphere. Histologic sections of the upper quadrant of the left and right hemispheres were immuno-stained with CD61 to visualize platelet aggregates in the microvasculature. Intravascular platelet aggregates (Figure 2A) were quantified using automated software analysis (Orbit) (CD61+ aggregates/mm2). Results All animals survived until the end of the procedure. Figure 1 shows differences in infarct size depending on the duration of occlusion (p=0.006). Figure 2 depicts an overall higher presence of platelet (CD61+) aggregates in the right hemisphere (infarct) compared to the left hemisphere (intact) (Figure 2B). There was a significant difference in platelet aggregates between left and right hemisphere but groupwise comparison showed only a trend (p=0.075) for more aggregates in the right hemisphere, compared to left, after 4 hours occlusion. Linear regression model could not identify whether infarct size and duration of occlusion were independent predictors of the number of platelet aggregates in the right hemisphere (p=0.191). Conclusions IMR occurs after brain ischemia in a swine model of AIS as a result of microvascular platelet aggregates. Further analysis and experiments are needed to elucidate which predictors can explain intravascular platelet aggregation.Infarct SizePlatelet aggregates
Mechanical load regulates the proliferation of multiple cell types in the heart
Authors: G Ciucci, A Colliva, S Vodret, B Texler, B Cardini, R Oberhuber, R Vuerich, E Zago, M Maglione, G Sinagra, M Giacca, T Eschenhagen, P Golino, F Loffredo, S Zacchigna
Journal: Cardiovascular Research (2024)

Abstract Funding Acknowledgements None. Introduction The adult mammalian heart has a poor regenerative and angiogenic potential. At the same time, both primary cardiac tumors and cardiac metastases are extremely rare. This suggests that shared mechanisms may halt the proliferation of cardiac (both cardiomyocyte and endothelial) and cancer cells. Among the mechanisms that have been claimed to be responsible for the loss of proliferative potential of cardiomyocytes early after birth is a sudden increase in mechanical load. Purpose Here, we investigate whether alterations in mechanical load affect the proliferation of multiple cell types in the heart. Methods Adult heart unloading in vivo was achieved using a mouse model of heterotopic heart transplantation, followed by EdU injection to label proliferating cells. Engineered heart tissues (EHTs) were generated using neonatal cardiomyocytes and fibroblasts, with a modified protocol allowing to finely tune mechanical load. Primary cardiac endothelial cells and multiple types of cancer cells (lung, melanoma and colon cancer) along with adult cardiac endothelial cells were included in EHTs. Results In line with our hypothesis, unloaded, heterotransplanted hearts showed a significant number of proliferating cardiomyocytes and endothelial cells, which were almost absent in native hearts. While cancer cells did not grow in native hearts, they massively proliferated in unloaded hearts. Consistent with in vivo results, mechanical unloading in EHTs significantly increased the percentage of EdU-positive cardiomyocytes, endothelial and cancer cells. In contrast, increasing afterload reduced cell proliferation in all cell types. Mechanistically, we identified Nesprin2 as an essential factor in sensing mechanical load and translating it into a cell proliferation arrest. Conclusions Overall, these data indicate that variations in mechanical load have a dramatic effect on the proliferation of multiple cell types within the heart tissue, including cardiomyocytes, endothelial and cancer cells and that Nesprin2 is required to mediate this effect.Unloading in the heartUnloading in EHT