scholarly journals Partial Inhibition of the 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase-3 (PFKFB3) Enzyme in Myeloid Cells Does Not Affect Atherosclerosis

2021 ◽  
Vol 9 ◽  
Author(s):  
Renée J. H. A. Tillie ◽  
Jenny De Bruijn ◽  
Javier Perales-Patón ◽  
Lieve Temmerman ◽  
Yanal Ghosheh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Myeloid Cells ◽  
Muscle Cells ◽  
Necrotic Core ◽  
Plaque Burden ◽  
Glycolytic Flux ◽  
Core Size ◽  
Plaque Stability ◽  
Positive Effects

BackgroundThe protein 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a key stimulator of glycolytic flux. Systemic, partial PFKFB3 inhibition previously decreased total plaque burden and increased plaque stability. However, it is unclear which cell type conferred these positive effects. Myeloid cells play an important role in atherogenesis, and mainly rely on glycolysis for energy supply. Thus, we studied whether myeloid inhibition of PFKFB3-mediated glycolysis in Ldlr–/–LysMCre+/–Pfkfb3fl/fl (Pfkfb3fl/fl) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr–/–LysMCre+/–Pfkfb3wt/wt control mice (Pfkfb3wt/wt).Methods and ResultsAnalysis of atherosclerotic human and murine single-cell populations confirmed PFKFB3/Pfkfb3 expression in myeloid cells, but also in lymphocytes, endothelial cells, fibroblasts and smooth muscle cells. Pfkfb3wt/wt and Pfkfb3fl/fl mice were fed a 0.25% cholesterol diet for 12 weeks. Pfkfb3fl/fl bone marrow-derived macrophages (BMDMs) showed 50% knockdown of Pfkfb3 mRNA. As expected based on partial glycolysis inhibition, extracellular acidification rate as a measure of glycolysis was partially reduced in Pfkfb3fl/fl compared to Pfkfb3wt/wt BMDMs. Unexpectedly, plaque and necrotic core size, as well as macrophage (MAC3), neutrophil (Ly6G) and collagen (Sirius Red) content were unchanged in advanced Pfkfb3fl/fl lesions. Similarly, early lesion plaque and necrotic core size and total plaque burden were unaffected.ConclusionPartial myeloid knockdown of PFKFB3 did not affect atherosclerosis development in advanced or early lesions. Previously reported positive effects of systemic, partial PFKFB3 inhibition on lesion stabilization, do not seem conferred by monocytes, macrophages or neutrophils. Instead, other Pfkfb3-expressing cells in atherosclerosis might be responsible, such as DCs, smooth muscle cells or fibroblasts.

scholarly journals Metformin Inhibits Vascular Calcification in Female Rat Aortic Smooth Muscle Cells via the AMPK-eNOS-NO Pathway

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3680-3689 ◽  
Author(s):  
Xiaorui Cao ◽  
Huan Li ◽  
Huiren Tao ◽  
Ning Wu ◽  
Lifeng Yu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Muscle Cells ◽  
Specific Marker ◽  
Female Rat ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Positive Effects ◽  
Compound C

Metformin exhibits diverse protective effects against diabetic complications, such as bone loss. Here, we investigated the effect of metformin on vascular calcification, another type 2 diabetes complication. In female rat aortic smooth muscle cells (RASMCs), we observed that metformin significantly alleviated β-glycerophosphate-induced Ca deposition and alkaline phosphatase activity, corresponding with reduced expression of some specific genes in osteoblast-like cells, including Runx2 and bone morphogenetic protein-2, and positive effects on α-actin expression, a specific marker of smooth muscle cells. Mechanistic analysis showed that phosphorylation levels of both AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) were increased with NO overproduction. After inhibition of either AMPK or eNOS with the pharmacologic inhibitors, compound C or Nω-Nitro-L-arginine methyl ester, NO production was lowered and metformin-meditated vascular protection against β-glycerophosphate-induced Ca deposition was removed. Our results support that metformin prevents vascular calcification via AMPK-eNOS-NO pathway.

scholarly journals Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics

2020 ◽  
Vol 127 (11) ◽  
pp. 1437-1455 ◽  
Author(s):  
Marie A.C. Depuydt ◽  
Koen H.M. Prange ◽  
Lotte Slenders ◽  
Tiit Örd ◽  
Danny Elbersen ◽  
...  
Keyword(s):  
T Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Cell ◽  
Association Studies ◽  
Myeloid Cells ◽  
Muscle Cells ◽  
Atherosclerotic Plaques ◽  
Genome Wide Association Studies ◽  
Mesenchymal Transition

Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. Objective: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. Methods and Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 + and CD8 + T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

scholarly journals Loss of PARP-1 attenuates diabetic arteriosclerotic calcification via Stat1/Runx2 axis

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Peng Li ◽  
Ying Wang ◽  
Xue Liu ◽  
Bin Liu ◽  
Zhao-yang Wang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Macrophage Polarization ◽  
Muscle Cells ◽  
Plaque Burden ◽  
Metabolic Complications ◽  
Runx2 Expression ◽  
Parp 1

AbstractAccelerated atherosclerotic calcification is responsible for plaque burden, especially in diabetes. The regulatory mechanism for atherosclerotic calcification in diabetes is poorly characterized. Here we show that deletion of PARP-1, a main enzyme in diverse metabolic complications, attenuates diabetic atherosclerotic calcification and decreases vessel stiffening in mice through Runx2 suppression. Specifically, PARP-1 deficiency reduces diabetic arteriosclerotic calcification by regulating Stat1-mediated synthetic phenotype switching of vascular smooth muscle cells and macrophage polarization. Meanwhile, both vascular smooth muscle cells and macrophages manifested osteogenic differentiation in osteogenic media, which was attenuated by PARP-1/Stat1 inhibition. Notably, Stat1 acts as a positive transcription factor by directly binding to the promoter of Runx2 and promoting atherosclerotic calcification in diabetes. Our results identify a new function of PARP-1, in which metabolism disturbance-related stimuli activate the Runx2 expression mediated by Stat1 transcription to facilitate diabetic arteriosclerotic calcification. PARP-1 inhibition may therefore represent a useful therapy for this challenging complication.

Abstract 17038: Twist1 Modulates Smooth Muscle Cell Phenotypes in Atherosclerosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Marie A Guerraty ◽  
Sylvia T Nurnberg ◽  
Vraj Shah ◽  
Daniel J Rader
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Risk Allele ◽  
Muscle Cells ◽  
Chromosome 9 ◽  
Alizarin Red ◽  
Plaque Stability ◽  
Atherosclerotic Lesions ◽  
Mesenchymal Transformation

Introduction: Genome-wide association studies have identified rs2107595, a non-coding locus on chromosome 9 between HDAC9 and Twist1 genes, as a risk allele for several vascular phenotypes, including Coronary Artery Disease (CAD). Rs2107595 has, more specifically, been associated with stable CAD over myocardial infarction phenotypes. Recent work has shown that rs2107595 risk allele increases Twist1 expression in smooth muscle cells (SMCs) by creating an RBPJ binding site. In other cell types, Twist1 is known to maintain cells in a de-differentiated state and to promote epithelial to mesenchymal transformation, driving tumor progression and metastasis. Hypothesis: Twist1 modulates SMC differentiation to promote an immature proliferative state over a differentiated (osteoblastic) state. This shift in phenotype promotes features of plaque stability in vivo . Methods: Twist1 expression plasmid (pCMV6-TWIST1) was transfected into A7r5 rat smooth muscle cells. To assess proliferation, cells were counted at 24, 48, 72, and 96 hours. To assess calcification, A7r5 cells were cultured in calcification media (2mM NaPhos) for 10 days and stained with Alizarin Red. In vivo studies were performed in Twist1 fl/fl tamoxifen-inducible MYH11-Cre C57BL/6 mice on ApoE-/- background fed a Western diet for 16 weeks to induce atherosclerotic lesions. Immunohistochemistry with SM22a identified lesion SMCs, and alizarin red was used to identify calcifications. Results: Ectopic overexpression of Twist1 in A7r5 SMCs decreased proliferation at 48h and 72h (80%, p=0.014). Twist 1 overexpression also decreased the total area of calcification (33% reduction, p=0.007). In vivo , both control and Twist 1 KO mice show similar burden of atherosclerosis. However, there is a decrease in sub-endothelial SMCs in atherosclerotic lesions by SM22a staining in the Twist1 KO. Additionally, Twist1 KO mice have more prominent and larger focal calcifications. Conclusions: Twist1 promotes SMC proliferation and decreases calcification in vitro , and may affect the presence of subendothelial SMCs and calcification in vivo . This provides a compelling link that rs2107595 may promote plaque stability in CAD by increasing Twist1 to modulate SMC phenotypes.

scholarly journals Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture

2021 ◽  
Vol 8 (5) ◽  
pp. 51
Author(s):  
Eoghan M. Cunnane ◽  
Aneesh K. Ramaswamy ◽  
Katherine L. Lorentz ◽  
David A. Vorp ◽  
Justin S. Weinbaum
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Stromal Cells ◽  
Biological Effects ◽  
Muscle Cells ◽  
Arterial Disease ◽  
Protein Quantification ◽  
Elastic Fibers ◽  
Positive Effects ◽  
Adipose Derived Stromal Cells

Macromolecular components of the vascular extracellular matrix (ECM), particularly elastic fibers and collagen fibers, are critical for the proper physiological function of arteries. When the unique biomechanical combination of these fibers is disrupted, or in the ultimate extreme where fibers are completely lost, arterial disease can emerge. Bioengineers in the realms of vascular tissue engineering and regenerative medicine must therefore ideally consider how to create tissue engineered vascular grafts containing the right balance of these fibers and how to develop regenerative treatments for situations such as an aneurysm where fibers have been lost. Previous work has demonstrated that the primary cells responsible for vascular ECM production during development, arterial smooth muscle cells (SMCs), can be induced to make new elastic fibers when exposed to secreted factors from adipose-derived stromal cells. To further dissect how this signal is transmitted, in this study, the factors were partitioned into extracellular vesicle (EV)-rich and EV-depleted fractions as well as unseparated controls. EVs were validated using electron microscopy, dynamic light scattering, and protein quantification before testing for biological effects on SMCs. In 2D culture, EVs promoted SMC proliferation and migration. After 30 days of 3D fibrin construct culture, EVs promoted SMC transcription of the elastic microfibril gene FBN1 as well as SMC deposition of insoluble elastin and collagen. Uniaxial biomechanical properties of strand fibrin constructs were no different after 30 days of EV treatment versus controls. In summary, it is apparent that some of the positive effects of adipose-derived stromal cells on SMC elastogenesis are mediated by EVs, indicating a potential use for these EVs in a regenerative therapy to restore the biomechanical function of vascular ECM in arterial disease.

Pathology of stable coronary artery disease

ESC CardioMed ◽  
2018 ◽  
pp. 1315-1320
Author(s):  
Michael Joner ◽  
Maria Isabel Castellanos ◽  
Anna Bulin ◽  
Kristin Steigerwald
Keyword(s):  
Coronary Artery Disease ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Necrotic Core ◽  
Intimal Thickening ◽  
Intraplaque Haemorrhage ◽  
Artery Disease ◽  
Plaque Destabilization

Coronary artery disease remains the major cause of morbidity and mortality on a global scale. Intimal thickening and fatty streaks represent early adaptive vascular changes, which are often regressive. Pathological intimal thickening represents the earliest progressive atherosclerotic lesion characterized by a focal accumulation of smooth muscle cells and acellular areas, often associated with lipid pools. Fibroatheroma is characterized by a necrotic core and can be split into early and late fibroatheroma, where macrophage apoptosis and defective efferocytosis play important roles in lesion progression. Intraplaque hypoxia is believed to result in neovascularization with subsequent intraplaque haemorrhage because of immature and leaky microvessels. Due to excessive cholesterol in remnants of erythrocyte membranes, intraplaque haemorrhage may result in rapid progression of necrotic core and plaque destabilization. Healed plaque rupture has been well delineated as an important mechanism of gradual luminal narrowing, where changes in collagen deposition allow recognition of rupture and healing sites. Calcification results from apoptosis of smooth muscle cells and macrophages or may also be caused by active release of cellular vesicles involved in calcium haemostasis. Extracellular matrix changes are associated with progression of atherosclerosis, while integrin signalling has been recognized as an important outside-in transcellular target of the inflammatory response.

Pathology of stable coronary artery disease

ESC CardioMed ◽  
2018 ◽  
pp. 1315-1320
Author(s):  
Michael Joner ◽  
Maria Isabel Castellanos ◽  
Anna Bulin ◽  
Kristin Steigerwald
Keyword(s):  
Coronary Artery Disease ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Necrotic Core ◽  
Intimal Thickening ◽  
Intraplaque Haemorrhage ◽  
Artery Disease ◽  
Plaque Destabilization

Coronary artery disease remains the major cause of morbidity and mortality on a global scale. Intimal thickening and fatty streaks represent early adaptive vascular changes, which are often regressive. Pathological intimal thickening represents the earliest progressive atherosclerotic lesion characterized by a focal accumulation of smooth muscle cells and acellular areas, often associated with lipid pools. Fibroatheroma is characterized by a necrotic core and can be split into early and late fibroatheroma, where macrophage apoptosis and defective efferocytosis play important roles in lesion progression. Intraplaque hypoxia is believed to result in neovascularization with subsequent intraplaque haemorrhage because of immature and leaky microvessels. Due to excessive cholesterol in remnants of erythrocyte membranes, intraplaque haemorrhage may result in rapid progression of necrotic core and plaque destabilization. Healed plaque rupture has been well delineated as an important mechanism of gradual luminal narrowing, where changes in collagen deposition allow recognition of rupture and healing sites. Calcification results from apoptosis of smooth muscle cells and macrophages or may also be caused by active release of cellular vesicles involved in calcium haemostasis. Extracellular matrix changes are associated with progression of atherosclerosis, while integrin signalling has been recognized as an important outside-in transcellular target of the inflammatory response.

Abstract 581: Lipid Peroxide Derived Dicarboxylic Acids Induce Atherosclerotic Calcification

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Aladdin Riad ◽  
Chandrakala A Narasimhulu ◽  
Dmitry Litvinov ◽  
Xueting Jiang ◽  
Irene F Ruiz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Azelaic Acid ◽  
Lipid Peroxide ◽  
Muscle Cells ◽  
The United States ◽  
Arterial Calcification ◽  
Plaque Burden ◽  
Alizarin Red ◽  
Ldl Particles

Background: Cardiovascular diseases, including atherosclerosis, are the leading cause of death in the United States. Atherosclerotic lesions are formed by deposition of lipids in the intima of arteries. Upon exposure to oxidative stresses, low-density lipoprotein (LDL) is converted to highly atherogenic oxidized LDL (ox-LDL) particles, which contribute to disease development and progression. Advanced disease stages may result in calcification of lesions. This calcification process is important, as it has been shown to be associated with stable plaques that are less prone to rupture. Calcification is present in lipid rich domains of lesions, however neither the composition of the mineralized calcium deposits nor its relationship to lipid peroxidation is known. Hypothesis: Lipid peroxide derived AZA induces calcification of smooth muscle cells thereby providing the link between calcification and overall plaque burden, and association of calcification with the lipophilic region of the lesion. Methods: In this study, the potential of lipid peroxide-derived lipophilic dicarboxylic acid (DCA, e.g. azelaic acid) to promote calcification upon exposure to vascular smooth muscle cells was tested. Using 13-Hydroperoxyoctadecadienoic acid (13-hydroperoxylinoleic acid, 13-HPODE) and thin-layer and gas chromatography[[Unable to Display Character: –]]mass spectrometry we characterized the conditions where HPODE is decomposed to aldehyde product 9-oxo-nonanoic acid and its corresponding DCA azelaic acid (AZA). Results: Both free AZA and intracellular delivery of AZA via lysophosphatidylcholine (lysoPtdCho) micelles induced calcification of aortic smooth muscle cells, as determined by Von Kossa and alizarin red staining. HPODE treatment resulted in the cellular conversion to ONA and AZA as determined by GC-MS. Conclusion: These results demonstrate that DCAs could contribute to atherosclerotic calcification thus accounting for the latter’s relationship to plaque burden and association with lipids. This study also challenges the dogma that arterial calcification represents the deposition of calcium phosphate. Our future work aims to delineate the association of calcium with lipid rich plaques and lipid oxidation with calcification in animal and human atherosclerosis.

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