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Objectives: We previously found that the pluripotency factor OCT4 is reactivated in smooth muscle cells (SMC) in human and mouse atherosclerotic plaques and plays an atheroprotective role. Loss of OCT4 in SMC in vitro was associated with decreases in SMC migration. However, molecular mechanisms responsible for atheroprotective SMC-OCT4-dependent effects remain unknown. Methods: Since studies in embryonic stem cells demonstrated that OCT4 regulates long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), making them candidates for OCT4 effect mediators, we applied an in vitro approach to investigate the interactions between OCT4-regulated lncRNAs, mRNAs, and miRNAs in SMC. We used OCT4 deficient mouse aortic SMC (MASMC) treated with the pro-atherogenic oxidized phospholipid POVPC, which, as we previously demonstrated, suppresses SMC contractile markers and induces SMC migration. Differential expression of lncRNAs, mRNAs, and miRNAs was obtained by lncRNA/mRNA expression array and small-RNA microarray. Long non-coding RNA to mRNA associations were predicted based on their genomic proximity and association with vascular diseases. Given a recently discovered crosstalk between miRNA and lncRNA, we also investigated the association of miRNAs with upregulated/downregulated lncRNA-mRNA pairs. Results: POVPC treatment in SMC resulted in upregulating genes related to the axon guidance and focal adhesion pathways. Knockdown of Oct4 resulted in differential regulation of pathways associated with phagocytosis. Importantly, these results were consistent with our data showing that OCT4 deficiency attenuated POVPC-induced SMC migration and led to increased phagocytosis. Next, we identified several up- or downregulated lncRNA associated with upregulation of the specific mRNA unique for the OCT4 deficient SMC, including upregulation of ENSMUST00000140952-Hoxb5/6 and ENSMUST00000155531-Zfp652 along with downregulation of ENSMUST00000173605-Parp9 and, ENSMUST00000137236-Zmym1. Finally, we found that many of the downregulated miRNAs were associated with cell migration, including miR-196a-1 and miR-10a, targets of upregulated ENSMUST00000140952, and miR-155 and miR-122, targets of upregulated ENSMUST00000155531. Oppositely, the upregulated miRNAs were anti-migratory and pro-phagocytic, such as miR-10a/b and miR-15a/b, targets of downregulated ENSMUST00000173605, and miR-146a/b and miR-15b targets of ENSMUST00000137236. Conclusion: Our integrative analyses of the lncRNA-miRNA-mRNA interactions in SMC indicated novel potential OCT4-dependent mechanisms that may play a role in SMC phenotypic transitions.
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BACKGROUND: Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. METHODS: We used human and mouse aortic EC as well as EC-lineage tracing (Cdh5-CreERT2 Rosa-YFP [yellow fluorescence protein]) atherosclerotic Apoe-/- mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. Bioinformatics analyses were performed on male and female mouse aortic EC and human lung and aortic EC. RESULTS: In vitro, female human and mouse aortic ECs showed more apoptosis and higher cellular reactive oxygen species levels than male EC. In addition, female mouse aortic EC had lower mitochondrial membrane potential (ΔΨm), lower TFAM (mitochondrial transcription factor A) levels, and decreased angiogenic potential (tube formation, cell viability, and proliferation) compared with male mouse aortic EC. In vivo, female mice had significantly higher lipid accumulation within the aortas, impaired glucose tolerance, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and endothelial-to-mesenchymal transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNA-seq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. CONCLUSIONS: Our study delineates critical sex-specific differences in EC relevant to proinflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biological basis for sex-specific proatherosclerotic mechanisms.
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Enfermedades de la Aorta , Aterosclerosis , Femenino , Masculino , Humanos , Ratones , Animales , Células Endoteliales/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/patología , Aorta/patología , Células Cultivadas , Especies Reactivas de Oxígeno/metabolismo , Inflamación/metabolismo , Ratones Endogámicos C57BLRESUMEN
Introduction: There is growing evidence that smooth muscle cell (SMC) phenotypic transitions play critical roles during normal developmental and tissue recovery processes and in pathological conditions such as atherosclerosis. However, the molecular mechanisms responsible for these transitions are not well understood. Recently, we found that the embryonic stem cell/induced pluripotent stem cell (iPSC) factor OCT4, which was believed to be silenced in somatic cells, plays an atheroprotective role in SMC, and regulates angiogenesis after corneal alkali burn and hindlimb ischemia by mediating microvascular SMC and pericyte migration. However, the kinetics of OCT4 activation in arterial SMC and its role in acute pathological conditions are still unknown. Methods and Results: Here, using an Oct4-IRES-GFP reporter mouse model, we found that OCT4 is reactivated in the carotid artery 18 hours post-acute ligation-induced injury, a common in vivo model of the SMC phenotypic transitions. Next, using a tamoxifen-inducible Myh11-CreERT2 Oct4 knockout mouse model, we found that the loss of OCT4, specifically in SMC, led to accelerated neointima formation and increased tunica media following carotid artery ligation, at least in part by increasing SMC proliferation within the media. Bulk RNA sequencing analysis on the cultured SMC revealed significant down-regulation of the SMC contractile markers and dysregulation of the genes belonging to the regulation of cell proliferation and, positive and negative regulation for cell migration ontological groups following genetic inactivation of Oct4. We also found that loss of Oct4 resulted in suppression of contractile SMC markers after the injury and in cultured aortic SMC. Further mechanistic studies revealed that OCT4 regulates SMC contractile genes, ACTA2 and TAGLN, at least in part by direct binding to the promoters of these genes. Conclusion: These results demonstrate that the pluripotency factor OCT4 is quickly activated in SMC after the acute vascular injury and inhibits SMC hyperproliferation, which may be protective in preventing excessive neointima formation.
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Despite many decades of research, complications of atherosclerosis resulting from the rupture or erosion of unstable plaques remain the leading cause of death worldwide. Advances in cellular lineage tracing techniques have allowed researchers to begin investigating the role of individual cell types in the key processes regulating plaque stability, including maintenance of the fibrous cap, a protective collagen-rich structure that underlies the endothelium. This structure was previously thought to be entirely derived from smooth muscle cells (SMC), which migrated from the vessel wall. However, recent lineage tracing studies have identified endothelial cells (EC) as an essential component of this protective barrier through an endothelial-to-mesenchymal transition (EndoMT), a process that has previously been implicated in pulmonary, cardiac, and kidney fibrosis. Although the presence of EndoMT in atherosclerotic plaques has been shown by several laboratories using EC-lineage tracing mouse models, whether EndoMT is detrimental (i.e., worsening disease progression) or beneficial (i.e., an athero-protective response that prevents plaque instability) remains uncertain as there are data to support both possibilities, which will be further discussed in this review.
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Aterosclerosis , Placa Aterosclerótica , Animales , Aterosclerosis/metabolismo , Colágeno/metabolismo , Células Endoteliales/metabolismo , Endotelio/metabolismo , Fibrosis , Ratones , Placa Aterosclerótica/patologíaRESUMEN
AIMS: Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis. METHODS AND RESULTS: Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3+ cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis. CONCLUSIONS: These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics.
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Aterosclerosis , Placa Aterosclerótica , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Linaje de la Célula , Humanos , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica/metabolismo , Transducción de SeñalRESUMEN
Stable atherosclerotic plaques are characterized by a thick, extracellular matrix-rich fibrous cap populated by protective ACTA2+ myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMCs). Herein, we show that in murine and human lesions, 20% to 40% of ACTA2+ fibrous cap cells, respectively, are derived from non-SMC sources, including endothelial cells (ECs) or macrophages that have undergone an endothelial-to-mesenchymal transition (EndoMT) or a macrophage-to-mesenchymal transition (MMT). In addition, we show that SMC-specific knockout of the Pdgfrb gene, which encodes platelet-derived growth factor receptor beta (PDGFRß), in Apoe-/- mice fed a Western diet for 18 weeks resulted in brachiocephalic artery lesions nearly devoid of SMCs but with no changes in lesion size, remodelling or indices of stability, including the percentage of ACTA2+ fibrous cap cells. However, prolonged Western diet feeding of SMC Pdgfrb-knockout mice resulted in reduced indices of stability, indicating that EndoMT- and MMT-derived MFs cannot compensate indefinitely for loss of SMC-derived MFs. Using single-cell and bulk RNA-sequencing analyses of the brachiocephalic artery region and in vitro models, we provide evidence that SMC-to-MF transitions are induced by PDGF and transforming growth factor-ß and dependent on aerobic glycolysis, while EndoMT is induced by interleukin-1ß and transforming growth factor-ß. Together, we provide evidence that the ACTA2+ fibrous cap originates from a tapestry of cell types, which transition to an MF-like state through distinct signalling pathways that are either dependent on or associated with extensive metabolic reprogramming.
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Metabolismo Energético/genética , Placa Aterosclerótica/patología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Actinas/metabolismo , Animales , Apolipoproteínas E/genética , Arteria Braquial/patología , Dieta Occidental , Células Endoteliales/metabolismo , Células Endoteliales/patología , Transición Epitelial-Mesenquimal , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/patología , Placa Aterosclerótica/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
OBJECTIVE: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-CreERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP+ cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP+; and unintended proinflammatory consequences. Using newly generated Myh11-DreERT2tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4Δ/Δ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1+ lymphatic endothelial cells in the epididymal AT. CONCLUSIONS: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.
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Tejido Adiposo/metabolismo , Plasticidad de la Célula , Factores de Transcripción de Tipo Kruppel/metabolismo , Miocitos del Músculo Liso/metabolismo , Obesidad/metabolismo , Paniculitis/metabolismo , Pericitos/metabolismo , Tejido Adiposo/patología , Animales , Glucemia/metabolismo , Linaje de la Célula , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Noqueados , Miocitos del Músculo Liso/patología , Obesidad/etiología , Obesidad/genética , Obesidad/patología , Paniculitis/etiología , Paniculitis/genética , Paniculitis/patología , Pericitos/patologíaRESUMEN
Major myeloid cell functions from adhesion to migration and phagocytosis are mediated by integrin adhesion complexes, also known as adhesome. The presence of a direct integrin binding partner Kindlin-3 is crucial for these functions, and its lack causes severe immunodeficiency in humans. However, how Kindlin-3 is incorporated into the adhesome and how its function is regulated is poorly understood. In this study, using nuclear magnetic resonance spectroscopy, we show that Kindlin-3 directly interacts with paxillin (PXN) and leupaxin (LPXN) via G43/L47 within its F0 domain. Surprisingly, disruption of Kindlin-3-PXN/LPXN interactions in Raw 264.7 macrophages promoted cell spreading and polarization, resulting in upregulation of both general cell motility and directed cell migration, which is in a drastic contrast to the consequences of Kindlin-3 knockout. Moreover, disruption of Kindlin-3-PXN/LPXN binding promoted the transition from mesenchymal to amoeboid mode of movement as well as augmented phagocytosis. Thus, these novel links between Kindlin-3 and key adhesome members PXN/LPXN limit myeloid cell motility and phagocytosis, thereby providing an important immune regulatory mechanism.
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Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Fagocitosis/fisiología , Animales , Sitios de Unión/fisiología , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Paxillin/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica/fisiología , Células RAW 264.7RESUMEN
OBJECTIVE: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic Apoe-/- mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of Apoe-/- mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of Apoe-/- mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in Apoe-/- mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, sIgM-/-Apoe-/-, mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age. CONCLUSIONS: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.
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Aterosclerosis/dietoterapia , Autoanticuerpos/inmunología , Dieta con Restricción de Grasas/métodos , Dieta Occidental , Inmunoglobulina M/inmunología , Animales , Apolipoproteínas E/metabolismo , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Masculino , Ratones , Oxidación-ReducciónRESUMEN
The stem cell pluripotency factor Oct4 serves a critical protective role during atherosclerotic plaque development by promoting smooth muscle cell (SMC) investment. Here, we show using Myh11-CreERT2 lineage-tracing with inducible SMC and pericyte (SMC-P) knockout of Oct4 that Oct4 regulates perivascular cell migration and recruitment during angiogenesis. Knockout of Oct4 in perivascular cells significantly impairs perivascular cell migration, increases perivascular cell death, delays endothelial cell migration, and promotes vascular leakage following corneal angiogenic stimulus. Knockout of Oct4 in perivascular cells also impairs perfusion recovery and decreases angiogenesis following hindlimb ischemia. Transcriptomic analyses demonstrate that expression of the migratory gene Slit3 is reduced following loss of Oct4 in cultured SMCs, and in Oct4-deficient perivascular cells in ischemic hindlimb muscle. Together, these results provide evidence that Oct4 plays an essential role within perivascular cells in injury- and hypoxia-induced angiogenesis.
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Neovascularización Fisiológica , Factor 3 de Transcripción de Unión a Octámeros/deficiencia , Células Madre Pluripotentes/metabolismo , Animales , Muerte Celular , Linaje de la Célula , Movimiento Celular , Células Cultivadas , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Femenino , Miembro Posterior , Isquemia/metabolismo , Isquemia/patología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/metabolismo , Neovascularización Patológica , Factor 3 de Transcripción de Unión a Octámeros/genética , Pericitos/metabolismo , Pericitos/patología , Células Madre Pluripotentes/patologíaRESUMEN
The long-term adverse effects of radiotherapy on cardiovascular disease are well documented. However, the underlying mechanisms responsible for this increased risk are poorly understood. Previous studies using rigorous smooth muscle cell (SMC) lineage tracing have shown abundant SMC investment into atherosclerotic lesions, where SMCs contribute to the formation of a protective fibrous cap. Studies herein tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do this, we exposed SMC lineage tracing (Myh11-ERT2Cre YFP+) mice to lethal radiation (1,200 cGy) followed by bone marrow transplantation prior to atherosclerosis development or vessel injury. Surprisingly, following irradiation, we observed a complete loss of SMC investment in 100% of brachiocephalic artery (BCA), carotid artery, and aortic arch lesions. Importantly, this was associated with a decrease in multiple indices of atherosclerotic lesion stability within the BCA. Interestingly, we observed anatomic heterogeneity, as SMCs accumulated normally into lesions of the aortic root and abdominal aorta, suggesting that SMC sensitivity to lethal irradiation occurs in blood vessels of neural crest origin. Taken together, these results reveal an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for cardiovascular disease.
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Aterosclerosis/patología , Tronco Braquiocefálico/efectos de la radiación , Músculo Liso Vascular/efectos de la radiación , Miocitos del Músculo Liso/efectos de la radiación , Animales , Aorta Abdominal/patología , Aorta Abdominal/efectos de la radiación , Aterosclerosis/etiología , Médula Ósea/efectos de la radiación , Trasplante de Médula Ósea , Tronco Braquiocefálico/patología , Diferenciación Celular/efectos de la radiación , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Noqueados para ApoE , Músculo Liso Vascular/citología , Irradiación Corporal TotalRESUMEN
A deeper understanding of the metastatic process is required for the development of new therapies that improve patient survival. Metastatic tumor cell growth and survival in distant organs is facilitated by the formation of a pre-metastatic niche that is composed of hematopoietic cells, stromal cells and extracellular matrix (ECM). Perivascular cells, including vascular smooth muscle cells (vSMCs) and pericytes, are involved in new vessel formation and in promoting stem cell maintenance and proliferation. Given the well-described plasticity of perivascular cells, we hypothesized that perivascular cells similarly regulate tumor cell fate at metastatic sites. We used perivascular-cell-specific and pericyte-specific lineage-tracing models to trace the fate of perivascular cells in the pre-metastatic and metastatic microenvironments. We show that perivascular cells lose the expression of traditional vSMC and pericyte markers in response to tumor-secreted factors and exhibit increased proliferation, migration and ECM synthesis. Increased expression of the pluripotency gene Klf4 in these phenotypically switched perivascular cells promoted a less differentiated state, characterized by enhanced ECM production, that established a pro-metastatic fibronectin-rich environment. Genetic inactivation of Klf4 in perivascular cells decreased formation of a pre-metastatic niche and metastasis. Our data revealed a previously unidentified role for perivascular cells in pre-metastatic niche formation and uncovered novel strategies for limiting metastasis.
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Plasticidad de la Célula/genética , Factores de Transcripción de Tipo Kruppel/genética , Miocitos del Músculo Liso/metabolismo , Metástasis de la Neoplasia/genética , Pericitos/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Técnicas In Vitro , Factor 4 Similar a Kruppel , Melanoma Experimental , Ratones , Músculo Liso Vascular/citología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Microambiente TumoralRESUMEN
Atherosclerotic plaque rupture with subsequent embolic events is a major cause of sudden death from myocardial infarction or stroke. Although smooth muscle cells (SMCs) produce and respond to collagens in vitro, there is no direct evidence in vivo that SMCs are a crucial source of collagens and that this impacts lesion development or fibrous cap formation. We sought to determine how conditional SMC-specific knockout of collagen type XV (COL15A1) in SMC lineage tracing mice affects advanced lesion formation given that 1) we have previously identified a Col15a1 sequence variant associated with age-related atherosclerosis, 2) COL15A1 is a matrix organizer enhancing tissue structural integrity, and 3) small interfering RNA-mediated Col15a1 knockdown increased migration and decreased proliferation of cultured human SMCs. We hypothesized that SMC-derived COL15A1 is critical in advanced lesions, specifically in fibrous cap formation. Surprisingly, we demonstrated that SMC-specific Col15a1 knockout mice fed a Western diet for 18 wk failed to form advanced lesions. SMC-specific Col15a1 knockout resulted in lesions reduced in size by 78%, with marked reductions in numbers and proliferating SMCs, and lacked a SMC and extracellular matrix-rich lesion or fibrous cap. In vivo RNA-seq analyses on SMC Col15a1 knockout and wild-type lesions suggested that a mechanism for these effects is through global repression of multiple proatherogenic inflammatory pathways involved in lesion development. These results provide the first direct evidence that a SMC-derived collagen, COL15A1, is critical during lesion pathogenesis, but, contrary to expectations, its loss resulted in marked attenuation rather than exacerbation of lesion pathogenesis.NEW & NOTEWORTHY We report the first direct in vivo evidence that a smooth muscle cell (SMC)-produced collagen, collagen type XV (COL15A1), is critical for atherosclerotic lesion development. SMC Col15a1 knockout markedly attenuated advanced lesion formation, likely through reducing SMC proliferation and impairing multiple proatherogenic inflammatory processes.
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Aterosclerosis/genética , Aterosclerosis/patología , Colágeno/genética , Miocitos del Músculo Liso/patología , Envejecimiento/patología , Animales , Aorta/citología , Linaje de la Célula , Dieta Aterogénica , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miografía , Rigidez VascularRESUMEN
Although somatic cell activation of the embryonic stem cell (ESC) pluripotency factor OCT4 has been reported, this previous work has been controversial and has not demonstrated a functional role for OCT4 in somatic cells. Here we demonstrate that smooth muscle cell (SMC)-specific conditional knockout of Oct4 in Apoe(-/-) mice resulted in increased lesion size and changes in lesion composition that are consistent with decreased plaque stability, including a thinner fibrous cap, increased necrotic core area, and increased intraplaque hemorrhage. Results of SMC-lineage-tracing studies showed that these effects were probably the result of marked reductions in SMC numbers within lesions and SMC investment within the fibrous cap, which may result from impaired SMC migration. The reactivation of Oct4 within SMCs was associated with hydroxymethylation of the Oct4 promoter and was hypoxia inducible factor-1α (HIF-1α, encoded by HIF1A) and Krüppel-like factor-4 (KLF4)-dependent. These results provide the first direct evidence that OCT4 has a functional role in somatic cells, and they highlight the potential role of OCT4 in normal and diseased somatic cells.
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Aterosclerosis/genética , Movimiento Celular/genética , Miocitos del Músculo Liso/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Placa Aterosclerótica/genética , Animales , Aorta/metabolismo , Apolipoproteínas E/genética , Western Blotting , Linaje de la Célula , Supervivencia Celular , Inmunoprecipitación de Cromatina , Enfermedad de la Arteria Coronaria/metabolismo , Dieta Occidental , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutagénesis Sitio-Dirigida , Miocitos del Músculo Liso/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using Myh11-CreER(T2) ROSA floxed STOP eYFP Apoe(-/-) mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Krüppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation-sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.
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Aterosclerosis/genética , Factores de Transcripción de Tipo Kruppel/genética , Miocitos del Músculo Liso/patología , Placa Aterosclerótica/genética , Animales , Apolipoproteínas E/antagonistas & inhibidores , Aterosclerosis/patología , Diferenciación Celular/genética , Linaje de la Célula , Rastreo Celular , Humanos , Factor 4 Similar a Kruppel , Macrófagos/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Placa Aterosclerótica/patología , Regiones Promotoras GenéticasRESUMEN
Smooth muscle cell (SMC) proliferation is a hallmark of vascular injury and disease. Global hypomethylation occurs during SMC proliferation in culture and in vivo during neointimal formation. Regardless of the programmed or stochastic nature of hypomethylation, identifying these changes is important in understanding vascular disease, as maintenance of a cells' epigenetic profile is essential for maintaining cellular phenotype. Global hypomethylation of proliferating aortic SMCs and concomitant decrease of DNMT1 expression were identified in culture during passage. An epigenome screen identified regions of the genome that were hypomethylated during proliferation and a region containing Collagen, type XV, alpha 1 (COL15A1) was selected by 'genomic convergence' for characterization. COL15A1 transcript and protein levels increased with passage-dependent decreases in DNA methylation and the transcript was sensitive to treatment with 5-Aza-2'-deoxycytidine, suggesting DNA methylation-mediated gene expression. Phenotypically, knockdown of COL15A1 increased SMC migration and decreased proliferation and Col15a1 expression was induced in an atherosclerotic lesion and localized to the atherosclerotic cap. A sequence variant in COL15A1 that is significantly associated with atherosclerosis (rs4142986, P = 0.017, OR = 1.434) was methylated and methylation of the risk allele correlated with decreased gene expression and increased atherosclerosis in human aorta. In summary, hypomethylation of COL15A1 occurs during SMC proliferation and the consequent increased gene expression may impact SMC phenotype and atherosclerosis formation. Hypomethylated genes, such as COL15A1, provide evidence for concomitant epigenetic regulation and genetic susceptibility, and define a class of causal targets that sit at the intersection of genetic and epigenetic predisposition in the etiology of complex disease.
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Aterosclerosis/genética , Senescencia Celular/genética , Colágeno/genética , Epigénesis Genética , Aterosclerosis/patología , Movimiento Celular/genética , Proliferación Celular , Células Cultivadas , Metilación de ADN/genética , Regulación de la Expresión Génica , Humanos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neointima/genéticaRESUMEN
Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a critical role in the development of atherosclerosis. However, the factors present within lesions that mediate VSMC phenotypic switching are unclear. Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), are active components of minimally modified low density lipoprotein and have been previously shown to induce multiple proatherogenic events in endothelial cells and macrophages, but their effects on VSMCs have been largely unexplored until recently. We previously showed that OxPLs induced phenotypic switching of VSMCs, including suppression of SMC differentiation marker genes. The goal of the present studies was to test the hypothesis that OxPLs alter extracellular matrix production and VSMC migration. Results showed that POVPC activated expression of several extracellular matrix proteins in VSMC. POVPC increased expression of type VIII collagen alpha1 chain (Col8a1) mRNA in cultured VSMCs and in vivo in rat carotid arteries by 9-fold and 4-fold, respectively. POVPC-induced activation of Col8a1 gene expression was reduced by small interfering RNA-mediated suppression of Krüppel-like factor 4 (Klf4) and Sp1, and was abolished in Klf4-knockout VSMCs. POVPC increased Klf4 binding to the Col8a1 gene promoter both in vivo in rat carotid arteries and in cultured VSMCs based on chromatin immunoprecipitation assays. Moreover, POVPC-induced VSMC migration was markedly reduced in Klf4- or type VIII collagen-knockout VSMCs. Given evidence that OxPLs are present within atherosclerotic lesions, it is interesting to suggest that OxPL-induced changes in VSMC phenotype may contribute to the pathogenesis of atherosclerosis at least in part through changes in extracellular matrix composition.
Asunto(s)
Movimiento Celular , Colágeno Tipo VIII/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Fosfolípidos/metabolismo , Animales , Aorta/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Arterias Carótidas/metabolismo , Células Cultivadas , Colágeno Tipo VIII/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Fenotipo , Fosfatidilcolinas/metabolismo , Éteres Fosfolípidos/metabolismo , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Factor de Transcripción Sp1/metabolismo , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
Atherosclerosis is a vascular disease characterized by lipid deposition and inflammation within the arterial wall. Oxidized phospholipids (oxPLs), such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) and its constituents 1-palmytoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are concentrated within atherosclerotic lesions and are known to be potent proinflammatory mediators. Phenotypic switching of smooth muscle cells (SMCs) plays a critical role in the development, progression, and end-stage clinical consequences of atherosclerosis, yet little is known regarding the effects of specific oxPLs on SMC phenotype. The present studies were focused on determining whether oxPLs regulate expression of SMC differentiation marker genes and the molecular mechanisms involved. Results showed that POVPC and PGPC induced profound suppression of smooth muscle (SM) alpha-actin and SM myosin heavy chain expression while simultaneously increasing expression of MCP-1, MCP-3, and cytolysin. OxPLs also induced nuclear translocation of Krüppel-like transcription factor 4 (KLF4), a known repressor of SMC marker genes. siRNA targeting of KLF4 nearly blocked POVPC-induced suppression of SMC marker genes, and myocardin. POVPC-induced repression of SMC marker genes was also significantly attenuated in KLF4 knockout SMCs. Taken together, these results suggest a novel role for oxPLs in phenotypic modulation of SMCs and indicate that these effects are dependent on the transcription factor, KLF4. These results may have important novel implications for the mechanisms by which oxPLs contribute to the pathogenesis of atherosclerosis.