bims-midhyp 2023-10-22 papers

bims-midhyp

Biomed News

on Mitochondrial dysfunction and hypoxia

Issue of 2023‒10‒22
twelve papers selected by
Alia Ablieh, Universität Heidelberg

The interplay between H19 and HIF-1α in mitochondrial dysfunction in myocardial infarction.
The metabolic control theory: Its development and its application to mitochondrial oxidative phosphorylation.
SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial-mediated fusion.
Transcriptome Analysis Reveals Molecular Signature and Cell-type Difference of Homo sapiens Endothelial-to-mesenchymal Transition.
Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells.
Mitochondrial Dynamics of Bcl-2 Family Proteins during 17-β-Estradiol-Induced Apoptosis Correlate with the Malignancy of Endometrial Cancer Cells.
HIF-1α regulates the cell viability in radioiodine-resistant papillary thyroid carcinoma cells induced by hypoxia through PKM2/NF-κB signaling pathway.
m6A demethylase FTO stabilizes LINK-A to exert oncogenic roles via MCM3-mediated cell-cycle progression and HIF-1α activation.
Novel functions of the ER-located Hsp40s DNAJB12 and DNAJB14 on proteins at the outer mitochondrial membrane under stress mediated by CCCP.
Transcriptomic analyses of juvenile Striped Bass (Morone saxatilis) exposed to chronic and acute temperature change.
Evaluation of Mitochondrial Function on Pyruvate Dehydrogenase Complex Deficient Patient-derived Cell Lines.
Protective Effect of MitoTEMPO Against Cardiac Dysfunction Caused by Ischemia-Reperfusion: MCAO Stroke Model Study.

Cell Signal. 2023 Oct 15. pii: S0898-6568(23)00334-0. [Epub ahead of print]112 110919

The interplay between H19 and HIF-1α in mitochondrial dysfunction in myocardial infarction.

Luhan Xie, Jiabei He, Jun Mao, Qingqing Zhang, Hongchen Bo, Lianhong Li.

  Myocardial infarction(MI) causes prolonged ischemia of infarcted myocardial tissue, which triggers a wide range of hypoxia cellular responses in cardiomyocytes. Emerging evidence has indicated the critical roles of long non-coding RNAs(lncRNAs) in cardiovascular diseases, including MI. The purpose of this study was to investigate the roles of lncRNA H19 and H19/HIF-1α pathway during MI. Results showed that cell injury and mitochondrial dysfunction were induced in hypoxia-treated H9c2 cells, accompanied by an increase in the expression of H19. H19 silencing remarkably diminishes cell injury, inhibits the dysfunctional degree of mitochondria, and decreases the injury of MI rats. Bioinformatics analysis and dual-luciferase assays revealed that H19 was the hypoxia-responsive lncRNA, and HIF-1α induced H19 transcription through direct binding to the H19 promoter. Moreover, H19 participates in the HIF-1α pathway by stabilizing the HIF-1α protein. These results indicated that H19 might be a potential biomarker and therapeutic target for myocardial infarction.

Keywords:  H19; HIF-1α; Hypoxia; Mitochondria; Myocardial infarction

DOI:  https://doi.org/10.1016/j.cellsig.2023.110919
Biosystems. 2023 Oct 12. pii: S0303-2647(23)00213-7. [Epub ahead of print] 105038

The metabolic control theory: Its development and its application to mitochondrial oxidative phosphorylation.

Jean-Pierre Mazat.

  Metabolic Control Theory (MCT) and Metabolic Control Analysis (MCA) are the two sides, theoretical and experimental, of the measurement of the sensitivity of metabolic networks in the vicinity of a steady state. We will describe the birth and the development of this theory from the first analyses of linear pathways up to a global mathematical theory applicable to any metabolic network. We will describe how the theory, given the global nature of mitochondrial oxidative phosphorylation, solved the problem of what controls mitochondrial ATP synthesis and then how it led to a better understanding of the differential tissue expression of human mitochondrial pathologies and of the heteroplasmy of mitochondrial DNA, leading to the concept of the threshold effect.

Keywords:  Metabolic control analysis; Metabolic control theory; Mitochondrial diseases; Oxidative phoshorylation

DOI:  https://doi.org/10.1016/j.biosystems.2023.105038
Cell Signal. 2023 Oct 17. pii: S0898-6568(23)00346-7. [Epub ahead of print] 110931

SIRT4 is a regulator of human skeletal muscle fatty acid metabolism influencing inner and outer mitochondrial-mediated fusion.

John Noone, Keith D Rochfort, Finbarr O'Sullivan, Donal J O'Gorman.

  OBJECTIVE: The mitochondrial phenotype, governed by the balance of fusion-fission, is a key determinant of energy metabolism. The inner and outer mitochondrial membrane (IMM) fusion proteins optic atrophy 1 (OPA1) and Mitofusin 1 and 2 (Mfn1/2) play an important role in this process. Recent evidence also shows that Sirtuin 4 (SIRT4), located within the mitochondria, is involved in the regulation of fatty acid oxidation. The purpose of this study was to determine if SIRT4 expression regulates inner and outer mitochondrial-mediated fusion and substrate utilization within differentiated human skeletal muscle cells (HSkMC).MATERIAL AND METHODS: SIRT4 expression was knocked down using small interfering RNA (siRNA) transfection in differentiated HSkMC. Following knockdown, mitochondrial respiration was determined by high-resolution respirometry (HRR) using the Oroboros Oxygraph O2k. Live cell confocal microscopy, quantified using the Mitochondrial Network Analysis (MiNA) toolset, was used to examine mitochondrial morphological change. This was further examined through the measurement of key metabolic and mitochondrial morphological regulators (mRNA and protein) induced by knockdown.
RESULTS: SIRT4 knockdown resulted in a significant decrease in LEAK respiration, potentially explained by a decrease in ANT1 protein expression. Knockdown further increased oxidative phosphorylation and protein expression of key regulators of fatty acid metabolism. Quantitative analysis of live confocal imaging of fluorescently labelled mitochondria following SIRT4 knockdown supported the role SIRT4 plays in the regulation of mitochondrial morphology, as emphasized by an increase in mitochondrial network branches and junctions. Measurement of key regulators of mitochondrial dynamics illustrated a significant increase in mitochondrial fusion proteins Mfn1, OPA1 respectively, indicative of an increase in mitochondrial size.
CONCLUSIONS: This study provides evidence of a direct relationship between the mitochondrial phenotype and substrate oxidation in HSkMC. We identify SIRT4 as a key protagonist of energy metabolism via its regulation of IMM and OMM fusion proteins, OPA1 and Mfn1. SIRT4 knockdown increases mitochondrial capacity to oxidize fatty acids, decreasing LEAK respiration and further increasing mitochondrial elongation via its regulation of mitochondrial fusion.

Keywords:  Metabolism; Mitochondrial dynamics; Mitochondrial function; OPA1; Sirtuin; Skeletal muscle

DOI:  https://doi.org/10.1016/j.cellsig.2023.110931
G3 (Bethesda). 2023 Oct 20. pii: jkad243. [Epub ahead of print]

Transcriptome Analysis Reveals Molecular Signature and Cell-type Difference of Homo sapiens Endothelial-to-mesenchymal Transition.

Ronald Bronson, Junfang Lyu, Jianhua Xiong.

  Endothelial-to-mesenchymal transition (EndoMT), a specific form of epithelial-to-mesenchymal transition, drives a growing number of human (Homo sapiens) pathological conditions. This emerging knowledge opens a path to discovering novel therapeutic targets for many EndoMT-associated disorders. Here, we constructed an atlas of the endothelial-cell transcriptome and demonstrated EndoMT-induced global changes in transcriptional gene expression. Our gene ontology analyses showed that EndoMT could be a specific checkpoint for leukocyte chemotaxis, adhesion, and transendothelial migration. We also identified distinct gene expression signatures underlying EndoMT across arterial, venous, and microvascular endothelial cells. We performed protein-protein interaction network analyses, identifying a class of highly connected hub genes in endothelial cells from different vascular beds. Moreover, we found that the short-chain fatty acid acetate strongly inhibits the transcriptional program of EndoMT in endothelial cells from different vascular beds across tissues. Our results reveal the molecular signature and cell-type difference of EndoMT across distinct tissue- and vascular-bed-specific endothelial cells, providing a powerful discovery tool and resource value. These results suggest that therapeutically manipulating the endothelial transcriptome could treat an increasing number of EndoMT-associated pathological conditions.

Keywords:   Homo sapiens endothelial cell; acetate; chemotaxis; endothelial-to-mesenchymal transition; transcriptome

DOI:  https://doi.org/10.1093/g3journal/jkad243
J Transl Med. 2023 Oct 17. 21(1): 729

Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells.

Jamie E Jeon, Lei Huang, Zhiyuan Zhu, Aaron Wong, Shaf Keshavjee, Mingyao Liu.

  BACKGROUND: Ischemia-reperfusion injury is a key complication following lung transplantation. The clinical application of ex vivo lung perfusion (EVLP) to assess donor lung function has significantly increased the utilization of "marginal" donor lungs with good clinical outcomes. The potential of EVLP on improving organ quality and ameliorating ischemia-reperfusion injury has been suggested.METHODS: To determine the effects of ischemia-reperfusion and EVLP on gene expression in human pulmonary microvascular endothelial cells and epithelial cells, cell culture models were used to simulate cold ischemia (4 °C for 18 h) followed by either warm reperfusion (DMEM + 10% FBS) or EVLP (acellular Steen solution) at 37 °C for 4 h. RNA samples were extracted for bulk RNA sequencing, and data were analyzed for significant differentially expressed genes and pathways.
RESULTS: Endothelial and epithelial cells showed significant changes in gene expressions after ischemia-reperfusion or EVLP. Ischemia-reperfusion models of both cell types showed upregulated pro-inflammatory and downregulated cell metabolism pathways. EVLP models, on the other hand, exhibited downregulation of cell metabolism, without any inflammatory signals.
CONCLUSION: The commonly used acellular EVLP perfusate, Steen solution, silenced the activation of pro-inflammatory signaling in both human lung endothelial and epithelial cells, potentially through the lack of serum components. This finding could establish the basic groundwork of studying the benefits of EVLP perfusate as seen from current clinical practice.

Keywords:  Cell culture model; Ex vivo organ perfusion; Ischemia reperfusion; Lung transplantation; RNA sequencing

DOI:  https://doi.org/10.1186/s12967-023-04601-w
Biochemistry. 2023 Oct 19.

Mitochondrial Dynamics of Bcl-2 Family Proteins during 17-β-Estradiol-Induced Apoptosis Correlate with the Malignancy of Endometrial Cancer Cells.

Takahiro Yaguchi, Misaki Kameno, Hirofumi Taira, Junji Kawakami.

Excessive fat intake leads to an increase in cholesterol. Overexposure to estrogen derived from cholesterol is known to contribute to the malignancy of endometrial adenocarcinomas. However, it is not well understood the relationship between the exposure to estrogen and the malignancy of endometrial adenocarcinomas. We investigated how estrogen affected the malignancy of endometrial cancer cells, specifically HEC1 cells (a moderately differentiated adenocarcinoma) and HEC50B cells (a poorly differentiated adenocarcinoma). Cell viability was decreased by exogenous 17-β-estradiol (E2) in a concentration-dependent manner. E2 disturbed the mitochondrial membrane potentials by changing the localization of the B-cell lymphoma 2 (Bcl-2) family protein; however, there were significant differences in the localization of Bcl-2 family proteins between HEC1 and HEC50B cells. In HEC1 cells, E2 increased the expression of B-cell lymphoma-extra large (Bcl-XL) and the Bcl-2-associated X protein (Bax) and decreased Bcl-2 and Bcl-2-associated death promoter (Bad) expression on the outer mitochondrial membrane. Conversely, E2 increased the expression of Bad and Bax, and it decreased Bcl-2 and Bcl-XL expressions on the outer mitochondrial membrane in HEC50B cells. The disturbance of the mitochondrial membrane potential led to the release of cytochrome c from the mitochondria to the cytosolic space followed by activating caspase-9. After that, caspase-3 was activated and induced apoptosis. These results suggested that the localization of the Bcl-2 family protein observed under E2-induced apoptosis is related to the malignancy of endometrial cancer cells. We hope that the dynamics of Bcl-2 family proteins such as Bcl-XL and Bad will be used to diagnose malignant endometrial adenocarcinomas.

DOI: https://doi.org/10.1021/acs.biochem.3c00064
Mol Carcinog. 2023 Oct 20.

HIF-1α regulates the cell viability in radioiodine-resistant papillary thyroid carcinoma cells induced by hypoxia through PKM2/NF-κB signaling pathway.

Dong Wang, Xiaoqian Liu, Meijing Li, Jinyao Ning.

  The curative treatment options for papillary thyroid cancer (PTC) encompass surgical intervention, radioactive iodine administration, and chemotherapy. However, the challenges of radioiodine (RAI) resistance, metastasis, and chemotherapy resistance remain inadequately addressed. The objective of this study was to investigate the protective role of hypoxia-inducible factor-1α (HIF-1α) in 131 I-resistant cells and a xenograft model under hypoxic conditions, as well as to explore potential mechanisms. The effects of HIF-1α on 131 I-resistant BCPAP and TPC-1 cells, as well as the xenograft model, were assessed in this study. Cell viability, migration, invasion, and apoptosis rates were measured using Cell Counting Kit-8, wound-healing, Transwell, and flow cytometry assays. Additionally, the expressions of Ki67, matrix metalloproteinase-9 (MMP-9), and pyruvate kinase M2 (PKM2) were examined using immunofluorescence or immunohistochemistry assays. Sodium iodide symporter and PKM2/NF-κBp65 relative protein levels were detected by western blot analysis. The findings of our study indicate that siHIF-1α effectively inhibits cell proliferation, cell migration, and invasion in 131 I-resistant cells under hypoxic conditions. Additionally, the treatment of siHIF-1α leads to alterations in the relative protein levels of Ki67, MMP-9, PKM2, and PKM2/NF-κBp65, both in vivo and in vitro. Notably, the effects of siHIF-1α are modified when DASA-58, an activator of PKM2, is administered. These results collectively demonstrate that siHIF-1α reduces cell viability in PTC cells and rat models, while also mediating the nuclear factor-κB (NF-κB)/PKM2 signaling pathway. Our findings provide a new rationale for further academic and clinical research on RAI-resistant PTC.

Keywords:  HIF-1α; cancer stem cells; hypoxic; papillary thyroid cancer; radioactive iodine

DOI:  https://doi.org/10.1002/mc.23648
Cell Rep. 2023 Oct 18. pii: S2211-1247(23)01285-8. [Epub ahead of print]42(10): 113273

m6A demethylase FTO stabilizes LINK-A to exert oncogenic roles via MCM3-mediated cell-cycle progression and HIF-1α activation.

Yabing Nan, Shi Liu, Qingyu Luo, Xiaowei Wu, Pengfei Zhao, Wan Chang, Ruixiang Zhang, Yin Li, Zhihua Liu.

  RNA N6-methyladenosine (m6A) modification is implicated in cancer progression, yet its role in regulating long noncoding RNAs during cancer progression remains unclear. Here, we report that the m6A demethylase fat mass and obesity-associated protein (FTO) stabilizes long intergenic noncoding RNA for kinase activation (LINK-A) to promote cell proliferation and chemoresistance in esophageal squamous cell carcinoma (ESCC). Mechanistically, LINK-A promotes the interaction between minichromosome maintenance complex component 3 (MCM3) and cyclin-dependent kinase 1 (CDK1), increasing MCM3 phosphorylation. This phosphorylation facilitates the loading of the MCM complex onto chromatin, which promotes cell-cycle progression and subsequent cell proliferation. Moreover, LINK-A disrupts the interaction between MCM3 and hypoxia-inducible factor 1α (HIF-1α), abrogating MCM3-mediated HIF-1α transcriptional repression and promoting glycolysis and chemoresistance. These results elucidate the mechanism by which FTO-stabilized LINK-A plays oncogenic roles and identify the FTO/LINK-A/MCM3/HIF-1α axis as a promising therapeutic target for ESCC.

Keywords:  CDK1; CP: Cancer; FTO; HIF-1α; LINK-A; MCM3; cell cycle progression; chemoresistance; esophageal squamous cell carcinoma; glycolysis; m(6)A

DOI:  https://doi.org/10.1016/j.celrep.2023.113273
Mol Cell Biochem. 2023 Oct 18.

Novel functions of the ER-located Hsp40s DNAJB12 and DNAJB14 on proteins at the outer mitochondrial membrane under stress mediated by CCCP.

Pattarawut Sopha, Tirawit Meerod, Bunkuea Chantrathonkul, Nadgrita Phutubtim, Douglas M Cyr, Piyarat Govitrapong.

  The endoplasmic reticulum (ER) membrane provides infrastructure for intracellular signaling, protein degradation, and communication among the ER lumen, cytosol, and nucleus via transmembrane and membrane-associated proteins. Failure to maintain homeostasis at the ER leads to deleterious conditions in humans, such as protein misfolding-related diseases and neurodegeneration. The ER transmembrane heat shock protein 40 (Hsp40) proteins, including DNAJB12 (JB12) and DNAJB14 (JB14), have been studied for their importance in multiple aspects of cellular events, including degradation of misfolded membrane proteins, proteasome-mediated control of proapoptotic Bcl-2 members, and assembly of multimeric ion channels. This study elucidates a novel facet of JB12 and JB14 in that their expression could be regulated in response to stress caused by the presence of ER stressors and the mitochondrial potential uncoupler CCCP. Furthermore, JB14 overexpression could affect the level of PTEN-induced kinase 1 (PINK1) expression under CCCP-mediated stress. Cells with genetic knockout (KO) of DNAJB12 and DNAJB14 exhibited an altered kinetic of phosphorylated Drp1 in response to the stress caused by CCCP treatment. Surprisingly, JB14-KO cells exhibited a prolonged stabilization of PINK1 during chronic exposure to CCCP. Cells depleted with JB12 or JB14 also revealed an increase in the mitochondrial count and branching. Hence, this study indicates the possible novel functions of JB12 and JB14 involving mitochondria in nonstress conditions and under stress caused by CCCP.

Keywords:  ER stress; Hsp40; Intracellular communication; J-protein; Mitochondrial dynamics; Mitochondrial stress response

DOI:  https://doi.org/10.1007/s11010-023-04866-1
PLoS One. 2023 ;18(10): e0289372

Transcriptomic analyses of juvenile Striped Bass (Morone saxatilis) exposed to chronic and acute temperature change.

Faith M Penny, Scott A Pavey.

Striped Bass are economically important, migratory fishes, which occur across a wide range of latitudes. Given their wide-ranging nature, Striped Bass can cope with a broad range of environmental temperatures, yet the mechanisms underlying this ability have not been thoroughly described. Heat shock proteins (HSPs) are inducible molecular chaperones, which help mitigate protein damage resulting from increased temperatures. The importance of HSPs has been demonstrated in a number of fish species, but their role in Striped Bass is poorly understood. This study characterizes changes in gene expression in juvenile Striped Bass, following acute and chronic temperature change. Fish were acclimated to one of three temperatures (15, 25 or 30°C) and sampled at one of two treatments (control or after CTmax), following which we assessed differential gene expression and gene ontology in muscle. It is clear from our differential expression analyses that acclimation to warm temperatures elicits more robust changes to gene expression, compared to acute temperature increases. Our differential expression analyses also revealed induction of many different heat shock proteins, including hsp70, hsp90, hsp40 and other small HSPs, after both acute and chronic temperature increase in white muscle. Furthermore, the most consistent gene ontology pattern that emerged following both acclimation and CTmax was upregulation of transcripts involved in "protein folding", which also include heat shock proteins. Gene ontology analyses also suggest changes to other processes after acclimation, including decreased growth pathways and changes to DNA methylation. Overall, these data suggest that HSPs likely play a major role in the Striped Bass's ability to tolerate warm waters.

DOI: https://doi.org/10.1371/journal.pone.0289372
Endocr Metab Immune Disord Drug Targets. 2023 Oct 11.

Evaluation of Mitochondrial Function on Pyruvate Dehydrogenase Complex Deficient Patient-derived Cell Lines.

Hana Pavlú-Pereira, Cristina Florindo, Filipa Carvalho, Isabel Tavares de Almeida, Joao Vicente, Vanessa Morais, Isabel Rivera.

  INTRODUCTION: Pyruvate Dehydrogenase Complex (PDC) is a pivotal gatekeeper between cytosolic glycolysis and mitochondrial oxidative phosphorylation, playing important role in aerobic energy metabolism. Most PDC deficiency, cases being caused by mutations in PDHA1 encoding the α subunit of the rate-limiting E1 enzyme, which is characterized by abnormal phenotypes caused by energy deprivation at peripheral/central nervous systems and muscular tissues. This study aims to evaluate the potential therapeutic effect of arginine and thiamine in ameliorating mitochondrial function in patient-derived cultured cells.MATERIALS AND METHODS: PDC-deficient cell lines, carrying three different PDHA1 variants, were cultured in the absence and presence of arginine and/or thiamine at therapeutical levels, 4 mM and 100 μM, respectively. Mitochondrial bioenergetics profile was evaluated using the Seahorse extracellular flux analyzer.
RESULTS: In physiological conditions, control cells presented standard values for all parameters evaluating the mitochondrial function, no differences being observed after supplementation of culture medium with therapeutic levels of arginine and/or thiamine. However, PDC-PDHA1 deficient cell lines consumed less oxygen than the control cells, but arginine and thiamine supplementation increased the basal respiration for values similar or higher than the control cell line. Moreover, arginine and thiamine treatment highlighted an inefficient oxidative phosphorylation carried out by PDC-deficient cell lines. Finally, this treatment showed an increased oxygen consumption by enzymes other than those in the respiratory chain, thus proving the dependence of these mutant cell lines on cytosolic sources for ATP production, namely glycolysis.
CONCLUSIONS: This study showed that arginine and thiamine, at therapeutical levels, increase the basal oxygen consumption rate of PDC-deficient cell lines, as well as their ATP-linked respiration. This parameter measures the capacity of the cell to meet its energetic demands and, therefore, its increase reveals a higher electron flow through the respiratory chain, which is coupled to elevated oxidative phosphorylation, thus indicating an overall increased robustness in mitochondrial- related bioenergetics.

Keywords:  arginine; energy metabolism; mitochondrial bioenergetics; pyruvate dehydrogenase complex; pyruvate dehydrogenase complex deficiency; thiamine

DOI:  https://doi.org/10.2174/0118715303280072231004082458
Int J Neurosci. 2023 Oct 20. 1-29

Protective Effect of MitoTEMPO Against Cardiac Dysfunction Caused by Ischemia-Reperfusion: MCAO Stroke Model Study.

Ahmet Akkoca, Belgin Büyükakilli, Ebru Balli, Burcu GültekiN, Erkan Özbay, Hatice Oruç Demirbağ, Çağatay Han Türkseven.

  PURPOSE: Neurological impairments are the leading cause of post-stroke mortality, while stroke-related cardiovascular diseases rank second in significance.This study investigates the potential protective effects of MitoTEMPO (2,2,6,6-tetramethyl-4-[[2-(triphenylphosphonio) acetyl] amino]-1-piperidinyloxy, monochloride, monohydrate), a mitochondria-specific antioxidant, against cardiac and neurological complications following stroke. The objective is to assess whether MitoTEMPO can be utilized as a protective agent for individuals with a high risk of stroke.MATERIALS AND METHODS: 17-week-old male Wistar Albino rats were randomly assigned to three groups: SHAM, ischemia-reperfusion (IR), and MitoTEMPO + ischemia-reperfusion (MT + IR; MitoTEMPO injection 0.7 mg/kg/day for 14 days). The SHAM group underwent a sham operation, while the IR group underwent 1-hour middle cerebral artery occlusion (MCAO) followed by 3 days of reperfusion. Afterwards, noninvasive thoracic electrical bioimpedance (TEB) and electrocardiography (ECG) measurements were taken, and sample collection was performed for histological and biochemical examinations.
RESULTS: Our TEB and ECG findings demonstrated that MitoTEMPO exhibited a protective effect on most parameters affected by IR compared to the SHAM group. Furthermore, our biochemical and histological data revealed a significant protective effect of MitoTEMPO against oxidative damage.
CONCLUSIONS: The findings suggest that both IR-induced cardiovascular abnormalities and the protective effect of MitoTEMPO may involve G-protein coupled receptor (GPCR)-mediated signaling mechanisms. This study was conducted with limitations including a single gender, a uniform age group, a specific stroke model limited to MCA, and pre-scheduled only one IR period. In future studies, addressing these limitations may enable the implementation of preventive measures for individuals at high risk of stroke.

Keywords:  Cardiovascular disease; Ischemic stroke; Middle cerebral artery occlusion

DOI:  https://doi.org/10.1080/00207454.2023.2273768