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BACKGROUND: The Global Iliac Branch Study (NCT05607277) is an international, multicenter, retrospective cohort study of anatomic predictors of adverse iliac events (AIEs) in aortoiliac aneurysms treated with iliac branch devices (IBDs). METHODS: Patients with pre-IBD and post-IBD computed tomography imaging were included. We measured arterial diameters, stenosis, calcification, bifurcation angles, and tortuosity indices using a standardized, validated protocol. A composite of ipsilateral AIE was defined, a priori, as occlusion, type I or III endoleak, device constriction, or clinical event requiring reintervention. Paired t-test compared tortuosity indices and splay angles pretreatment and post-treatment for all IBDs and by device material (stainless steel and nitinol). Two-sample t-test compared anatomical changes from pretreatment to post-treatment by device material. Logistic regression assessed associations between AIE and anatomic measurements. Analysis was performed by IBD. RESULTS: We analyzed 297 patients (286 males, 11 females) with 331 IBDs (227 stainless steel, 104 nitinol). Median clinical follow-up was 3.8 years. Iliac anatomy was significantly straightened with all IBD treatment, though stainless steel IBDs had a greater reduction in total iliac artery tortuosity index and aortic splay angle compared to nitinol IBDs (absolute reduction -0.20 [-0.22 to -0.18] vs. -0.09 [-0.12 to -0.06], P < 0.0001 and -19.6° [-22.4° to -16.9°] vs. -11.2° [-15.3° to -7.0°], P = 0.001, respectively). There were 54 AIEs in 44 IBDs in 42 patients (AIE in 13.3% of IBD systems), requiring 35 reinterventions (median time to event 41 days; median time to reintervention 153 days). There were 18 endoleaks, 29 occlusions, and 5 device constrictions. There were no strong associations between anatomic measurements and AIE overall, though internal iliac diameter was inversely associated with AIE in nitinol devices (nAIE, nitinol = 8). CONCLUSIONS: Purpose-built IBDs effectively treat aortoiliac disease, including that with tortuous anatomy, with a high patency rate (91.5%) and low reintervention rate (9.1%) at 4 years. Anatomic predictors of AIE are limited.
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OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease characterized by smooth muscle cell depletion, ECM (extracellular matrix) degradation, and infiltration of immune cells. The cellular and molecular profiles that govern the heterogeneity of the AAA aorta are yet to be elucidated. Approach and Results: We performed single-cell RNA sequencing on mouse AAA tissues. AAA was induced in C57BL/6J mice by perivascular application of CaCl2. Unbiased clustering identified 12 distinct populations of 8 cell types. Percentages of each population and gene expression were compared between sham and AAA tissue. Furthermore, we characterized the transcriptional profiles and potential functional features of populations in smooth muscle cells, fibroblasts, and macrophages and revealed the unique regulons in each cell type. CONCLUSIONS: Together, these data provide high-resolution insight into the complexity and heterogeneity of mouse AAA and indicate that populations within major cell types such as smooth muscle cells, fibroblasts, and macrophages may contribute differently to AAA pathogenesis. Graphic Abstract: A graphic abstract is available for this article.
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Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/etiologia , Proliferação de Células , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , RNA-Seq , Análise de Célula ÚnicaRESUMO
Deep vein thrombosis (DVT) is a common cardiovascular disease with a major effect on quality of life, and safe and effective therapeutic measures to efficiently reduce existent thrombus burden are scarce. Using a comprehensive targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics approach, we established temporal clusters of endogenously biosynthesized specialized proresolving mediators (SPMs) and proinflammatory and prothrombotic lipid mediators during DVT progression in mice. Administration of resolvin D4 (RvD4), an SPM that was enriched at the natural onset of thrombus resolution, significantly reduced thrombus burden, with significantly less neutrophil infiltration and more proresolving monocytes in the thrombus, as well as an increased number of cells in an early apoptosis state. Moreover, RvD4 promoted the biosynthesis of other D-series resolvins involved in facilitating resolution of inflammation. Neutrophils from RvD4-treated mice were less susceptible to an ionomycin-induced release of neutrophil extracellular traps (NETs), a meshwork of decondensed chromatin lined with histones and neutrophil proteins critical for DVT development. These results suggest that delivery of SPMs, specifically RvD4, modulates the severity of thrombo-inflammatory disease in vivo and improves thrombus resolution.
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Ácidos Graxos Insaturados/uso terapêutico , Trombose Venosa/tratamento farmacológico , Animais , Progressão da Doença , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/imunologia , Lipídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Trombose Venosa/imunologia , Trombose Venosa/patologiaRESUMO
OBJECTIVE: The GORE EXCLUDER iliac branch endoprosthesis (IBE; W.L. Gore & Associates, Flagstaff, Ariz) is designed to preserve internal iliac artery (IIA) patency during endovascular treatment of aneurysms involving the common iliac artery. The device is intended to conform to iliac tortuosity, which may decrease adverse iliac events (AIE). The objective of this study was to evaluate risk factors for AIE after IBE implantation. METHODS: This was a post hoc analysis of the prospective, multicenter GORE 12-04 IBE pivotal trial. Patients with preoperative and postoperative axial imaging were included, with analysis based on each treated iliac system. An independent core laboratory performed all scan measurements, including iliac diameters, lengths, and tortuosity. Conformability was analyzed by the changes in tortuosity after IBE deployment, with less change indicating greater conformation. The end point was AIE, defined as ipsilateral radiographic or clinical complications. Critical nonconformation was defined as a threshold change in tortuosity associated with a significant increase in AIE. RESULTS: We included 98 patients with 101 treated iliac systems. There were eight AIE (8%; six IIA component occlusions, one iliac branch component occlusion, and one EIA dissection requiring reintervention). Patients with AIE had smaller IIA diameters and less IBE conformability. After multivariable logistic regression analysis, an IIA diameter of less than 10 mm and a change in total iliac tortuosity beyond -15% were independently associated with AIE (odds ratio, 12 [interquartile range, 1.4-110] and odds ratio, 8.2 [interquartile range, 1.5-46], respectively), and the latter was used to define critical nonconformation. Critical nonconformation occurred in 11% of treated systems, and was associated with a high rate of AIE (36% vs 4%; P = .004). CONCLUSIONS: Endograft conformation is a novel device property and technical outcome that, along with a larger IIA diameter, is associated with freedom from AIE after IBE deployment. An evaluation of these risk factors may better inform the management of patients with iliac aneurysmal disease. Further research on endograft conformation and patient outcomes is warranted, particularly for those with challenging anatomy undergoing complex procedures.
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Implante de Prótese Vascular/instrumentação , Prótese Vascular , Procedimentos Endovasculares/instrumentação , Aneurisma Ilíaco/cirurgia , Artéria Ilíaca/cirurgia , Idoso , Idoso de 80 Anos ou mais , Implante de Prótese Vascular/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Feminino , Humanos , Aneurisma Ilíaco/diagnóstico por imagem , Aneurisma Ilíaco/fisiopatologia , Artéria Ilíaca/diagnóstico por imagem , Artéria Ilíaca/fisiopatologia , Masculino , Pessoa de Meia-Idade , Desenho de Prótese , Fatores de Tempo , Resultado do Tratamento , Estados Unidos , Grau de Desobstrução VascularRESUMO
Cardiovascular diseases, including peripheral arterial and venous disease, myocardial infarction, and stroke, are the number one cause of death worldwide annually. In the last 20 years, the role of necroptosis, a newly identified form of regulated necrotic cell death, in cardiovascular disease has come to light. Specifically, the damaging role of two kinase proteins pivotal in the necroptosis pathway, Receptor Interacting Protein Kinase 1 (RIPK1) and Receptor Interacting Protein Kinase 3 (RIPK3), in cardiovascular disease has become a subject of great interest and importance. In this review, we provide an overview of the current evidence supporting a pathologic role of RIPK1 and RIPK3 in cardiovascular disease. Moreover, we highlight the evidence behind the efficacy of targeted RIPK1 and RIPK3 inhibitors in the prevention and treatment of cardiovascular disease.
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Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Humanos , Necroptose , Transdução de SinaisRESUMO
Galectin-3-binding protein (gal3bp) and its receptor/ligand, galectin-3 (gal3), are secreted proteins that initiate signaling cascades in several diseases, and recent human proteomic data suggest they may play a role in venous thrombosis (VT). We hypothesized that gal3bp and gal3 may promote VT. Using a mouse stasis model of VT, we found that gal3bp and gal3 were localized on vein wall, red blood cells, platelets, and microparticles, whereas leukocytes expressed gal3 only. Gal3 was dramatically increased during early VT and gal3bp:gal3 colocalized in the leukocyte/endothelial cell interface, where leukocytes were partially attached to the vein wall. Thrombus size correlated with elevated gal3 and interleukin-6 (IL-6) vein wall levels. Recombinant gal3 promoted VT and increased vein wall IL-6 mRNA. Although recombinant gal3 restored the VT size in gal3(-/-) mice, it had no effect on IL6(-/-) mice, suggesting that gal3:gal3bp promotes VT through IL-6. Moreover, significantly fewer activated neutrophils were present in the gal3(-/-) vein walls. In a group of human patients, elevated circulating gal3bp correlated with acute VT. In conclusion, gal3bp:gal3 play a critical role in VT, likely via IL-6 and PMN-mediated thrombotic mechanisms, and may be a potential biomarker in human VT.
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Galectina 3/metabolismo , Glicoproteínas/metabolismo , Trombose Venosa/metabolismo , Animais , Antígenos de Neoplasias/sangue , Biomarcadores/sangue , Biomarcadores Tumorais/sangue , Plaquetas/metabolismo , Proteínas de Transporte/sangue , Movimento Celular , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Galectina 3/deficiência , Galectina 3/genética , Glicoproteínas/sangue , Humanos , Interleucina-6/deficiência , Interleucina-6/genética , Interleucina-6/metabolismo , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombose Venosa/sangue , Trombose Venosa/etiologiaRESUMO
BACKGROUND: Brachial-cephalic arteriovenous fistulas (BCFs) are associated with high-flow volumes, leading to potential risks such as arm swelling, steal syndrome, pseudoaneurysm (due to a pressurized access), and cephalic arch stenosis. We hypothesized that a proximal radial-cephalic fistula (prRCF) configuration mitigates these risks because a lower flow state is created. Furthermore, we also hypothesized that despite these lower flows, patencies (primary, primary assisted, secondary) are sustained. METHODS: Leveraging a prospectively collected database supplemented with detailed medical record data, analyses of patients undergoing BCF and prRCF were completed (November 2008 through March 2016). Preoperative clinical and imaging characteristics, operative variables, and postoperative complications were reviewed. The primary end point was a composite of arm swelling, steal, and pseudoaneurysm at 2 years. Fistulograms and interventions (surgical revision, thrombectomy, endovascular treatment of cephalic arch stenosis) censored at 2 years were compared between configurations. Patencies were plotted using Kaplan-Meier techniques and compared using Cox proportional hazards. RESULTS: During the study period, 345 arteriovenous fistulas and 72 prosthetic grafts were primarily placed; 56 patients underwent BCF and 50 patients underwent prRCF with a mean follow-up of 1.8 ± 1.7 (standard deviation) years. Except for prRCF patients being older, there was no difference between the groups with regard to preoperative characteristics. The artery diameter used for anastomosis was significantly larger in the BCF group (4.0 ± 1.1 mm vs 2.6 ± 0.8 mm; P < .001), with higher flow volumes at 6-week ultrasound examination (1060 ± 587 mL/min vs 735 ± 344 mL/min; P < .001). Complications (arm swelling, steal, pseudoaneurysm) were significantly more common in the BCF group (P = .02). There was a trend, albeit statistically insignificant, for the BCF group to require more cephalic arch stenosis interventions. Of those patients needing dialysis within 1 year, both BCF and prRCF were successfully used in the majority of patients (n = 27 [66%] vs n = 25 [63%]; P = 1.0). Unadjusted and adjusted primary, primary assisted, and secondary patency rates were similar between the groups. CONCLUSIONS: prRCFs have fewer complications yet similar midterm durability compared with BCFs. When it is anatomically feasible, prRCFs should be constructed over BCFs because of their superior physiology and clinical outcomes.
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Derivação Arteriovenosa Cirúrgica/métodos , Implante de Prótese Vascular/métodos , Artéria Braquial/cirurgia , Artéria Radial/cirurgia , Diálise Renal , Extremidade Superior/irrigação sanguínea , Grau de Desobstrução Vascular , Veias/cirurgia , Idoso , Idoso de 80 Anos ou mais , Falso Aneurisma/etiologia , Falso Aneurisma/fisiopatologia , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Velocidade do Fluxo Sanguíneo , Implante de Prótese Vascular/efeitos adversos , Artéria Braquial/diagnóstico por imagem , Artéria Braquial/fisiopatologia , Distribuição de Qui-Quadrado , Bases de Dados Factuais , Edema/etiologia , Edema/fisiopatologia , Feminino , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Isquemia/etiologia , Isquemia/fisiopatologia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Artéria Radial/diagnóstico por imagem , Artéria Radial/fisiopatologia , Fluxo Sanguíneo Regional , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Veias/diagnóstico por imagem , Veias/fisiopatologiaRESUMO
BACKGROUND: Autophagy is a major cellular process by which cytoplasmic components such as damaged organelles and misfolded proteins are recycled. Although low levels of autophagy occur in cells under basal conditions, certain cellular stresses including nutrient depletion, DNA damage, and oxidative stress are known to robustly induce autophagy. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor activated during oxidative stress to maintain genomic stability. Both autophagy and KLF4 play important roles in response to stress and function in tumor suppression. METHODS: To explore the role of KLF4 on autophagy in mouse embryonic fibroblasts (MEFs), we compared wild-type with Klf4 deficient cells. To determine the levels of autophagy, we starved MEFs for different times with Earle's balanced salts solution (EBSS). Rapamycin was used to manipulate mTOR activity and autophagy. The percentage of cells with γ-H2AX foci, a marker for DNA damage, and punctate pattern of GFP-LC3 were counted by confocal microscopy. The effects of the drug treatments, Klf4 overexpression, or Klf4 transient silencing on autophagy were analyzed using Western blot. Trypan Blue assay and flow cytometry were used to study cell viability and apoptosis, respectively. qPCR was also used to assay basal and the effects of Klf4 overexpression on Atg7 expression levels. RESULTS: Here our data suggested that Klf4 (-/-) MEFs exhibited impaired autophagy, which sensitized them to cell death under nutrient deprivation. Secondly, DNA damage in Klf4-null MEFs increased after treatment with EBSS and was correlated with increased apoptosis. Thirdly, we found that Klf4 (-/-) MEFs showed hyperactive mTOR activity. Furthermore, we demonstrated that rapamycin reduced the increased level of mTOR in Klf4 (-/-) MEFs, but did not restore the level of autophagy. Finally, re-expression of Klf4 in Klf4 deficient MEFs resulted in increased levels of LC3II, a marker for autophagy, and Atg7 expression level when compared to GFP-control transfected Klf4 (-/-) MEFs. CONCLUSION: Taken together, our results strongly suggest that KLF4 plays a critical role in the regulation of autophagy and suppression of mTOR activity. In addition, we showed that rapamycin decreased the level of mTOR in Klf4 (-/-) MEFs, but did not restore autophagy. This suggests that KLF4 regulates autophagy through both mTOR-dependent and independent mechanisms. Furthermore, for the first time, our findings provide novel insights into the mechanism by which KLF4 perhaps prevents DNA damage and apoptosis through activation of autophagy.
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Apoptose , Autofagia , Dano ao DNA , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Fator 4 Semelhante a Kruppel , Camundongos Endogâmicos C57BL , Modelos Biológicos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVE: To determine whether F-fluorodeoxyglucose (F-FDG) micro-positron emission tomography (micro-PET) can predict abdominal aortic aneurysm (AAA) rupture. BACKGROUND: An infrarenal AAA model is needed to study inflammatory mechanisms that drive rupture. F-FDG PET can detect vascular inflammation in animal models and patients. METHODS: After exposing Sprague-Dawley rats to intra-aortic porcine pancreatic elastase (PPE) (12 U/mL), AAA rupture was induced by daily, subcutaneous, ß-aminopropionitrile (BAPN, 300 mg/kg, N = 24) administration. Negative control AAA animals (N = 15) underwent daily saline subcutaneous injection after PPE exposure. BAPN-exposed animals that did not rupture served as positive controls [nonruptured AAA (NRAAA) 14d, N = 9]. Rupture was witnessed using radiotelemetry. Maximum standard uptakes for F-FDG micro-PET studies were determined. Aortic wall PAI-1, uPA, and tPA concentrations were determined by western blot analyses. Interleukin (IL)-1ß, IL-6, IL-10, and MIP-2 were determined by Bio-Plex bead array. Neutrophil and macrophage populations per high-power field were quantified. Matrix metalloproteinase (MMP) activities were determined by zymography. RESULTS: When comparing ruptured AAA (RAAA) to NRAAA 14d animals, increased focal F-FDG uptakes were detected at subsequent sites of rupture (P = 0.03). PAI-1 expression was significantly less in RAAA tissue (P = 0.01), with comparable uPA and decreased tPA levels (P = 0.02). IL-1ß (P = 0.04), IL-6 (P = 0.001), IL-10 (P = 0.04), and MIP-2 (P = 0.02) expression, neutrophil (P = 0.02) and macrophage presence (P = 0.002), and MMP9 (P < 0.0001) activity were increased in RAAA tissue. CONCLUSIONS: With this AAA rupture model, increased prerupture F-FDG uptake on micro-PET imaging was associated with increased inflammation in the ruptured AAA wall. F-FDG PET imaging may be used to monitor inflammatory changes before AAA rupture.
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Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Ruptura Aórtica/diagnóstico por imagem , Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Aminopropionitrilo , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Ruptura Aórtica/induzido quimicamente , Ruptura Aórtica/metabolismo , Ruptura Aórtica/patologia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Masculino , Tomografia por Emissão de Pósitrons/métodos , Ratos , Ratos Sprague-DawleyRESUMO
The transport of proteins between the cytoplasm and nucleus requires interactions between soluble transport receptors (karyopherins) and phenylalanine-glycine (FG) repeat domains on nuclear pore complex proteins (nucleoporins). However, the role of specific FG repeat-containing nucleoporins in nuclear protein export has not been carefully investigated. We have developed a novel kinetic assay to investigate the relative export kinetics mediated by the karyopherin Msn5/Kap142 in yeast containing specific FG-Nup mutations. Using the Msn5 substrate Crz1 as a marker for Msn5-mediated protein export, we observe that deletions of NUP100 or NUP2 result in decreased rates of Crz1 export, while nup60Δ and nup42Δ mutants do not vary significantly from wild type. The decreased Msn5 export rate in nup100Δ was confirmed using Mig1-GFP as a transport substrate. A nup100ΔGLFG mutant shows defects in nuclear export kinetics similar to a nup100Δ deletion. Removal of FG-repeats from Nsp1 also decreases export kinetics, while a loss of Nup1 FXFGs does not. To confirm that our export data reflected functional differences in protein localization, we performed Crz1 transcription activation assays using a CDRE::LacZ reporter gene that is upregulated upon increased transcription activation by Crz1 in vivo. We observe that expression from this reporter increases in nup100ΔGLFG and nsp1ΔFGΔFXFG strains that exhibit decreased Crz1 export kinetics but resembles wild-type levels in nup1ΔFXFG strains that do not exhibit export defects. These data provide evidence that the export of Msn5 is likely mediated by a specific subset of FG-Nups and that the GLFG repeat domain of Nup100 is important for Msn5-mediated nuclear protein export.
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Transporte Ativo do Núcleo Celular/genética , Carioferinas , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sequência de Aminoácidos/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicina/genética , Carioferinas/genética , Carioferinas/metabolismo , Mutação , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Fenilalanina/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Importance: Childbearing has been a particular barrier to successful recruitment and retention of women in surgery. Pregnant surgeons are more likely to have major pregnancy complications, such as preterm delivery, intrauterine growth restriction, infertility, and miscarriage, compared with nonsurgeons. The average obstetric complication rate for surgeons ranges between 25% and 82% in the literature and is considerably higher than that in the general US population at 5% to 15%. Observations: The risks that pregnant surgeons experience were individually analyzed. These risks included missed prenatal care; musculoskeletal hazards, such as prolonged standing, lifting, and bending; long work hours; overnight calls; exposure to teratogenic agents, such as ionizing radiation, anesthetic gases, chemotherapy agents, and methyl methacrylate; and psychological stress and discrimination from the long-standing stigma associated with balancing motherhood and professional life. Conclusions and Relevance: A clear, translatable, and enforceable policy addressing perinatal care of surgeons was proposed, citing evidence of the risks reviewed from the literature. A framework of protection for pregnant individuals is essential for attracting talented students into surgery, retaining talented surgical trainees and faculty, and protecting pregnant surgeons and their fetuses.
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Deep vein thrombosis (DVT) is a common clinical problem, but its cellular and molecular mechanisms remain incompletely understood. In this study, we performed single-cell RNA sequencing on mouse inferior vena cava (IVC) 24 h after thrombus-inducing IVC ligation or sham operation. 9 cell types composed of multiple subpopulations were identified. Notable transcriptomic changes induced by DVT included a marked inflammatory response, elevated hypoxia, and globally reduced myogenesis. Analysis of individual cell populations revealed increased inflammation and reduced extracellular matrix production across smooth muscle cells and fibroblasts, juxtaposed against an early phenotypic shift in smooth muscle cell populations away from a contractile state. By characterizing the transcriptomic changes in the vein wall during acute venous thrombosis at the single-cell level, this work provides novel insights into early pathological events in the vein wall that may potentiate thrombus formation and result in long term adverse venous remodeling.
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Trombose Venosa , Animais , Camundongos , Matriz Extracelular , Análise de Sequência de RNA , Veia Cava Inferior , Trombose Venosa/genéticaRESUMO
Cell-cell communication coordinates cellular differentiation, tissue homeostasis, and immune responses in states of health and disease. In abdominal aortic aneurysm (AAA), a relatively common and potentially life-threatening vascular disease, intercellular communications between multiple cell types are not fully understood. In this study, we analyzed published single-cell RNA sequencing (scRNA-seq) datasets generated from the murine CaCl2 model, perivascular elastase model, Angiotensin II model, and human AAA using bioinformatic approaches. We inferred the intercellular communication network in each experimental AAA model and human AAA and predicted commonly altered signaling pathways, paying particular attention to thrombospondin (THBS) signaling between different cell populations. Together, our analysis inferred intercellular signaling in AAA based on single-cell transcriptomics. This work provides important insight into cell-cell communications in AAA and has laid the groundwork for future experimental investigations that can elucidate the cell signaling pathways driving AAA.
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Abdominal aortic aneurysm (AAA), defined as a focal dilation of the abdominal aorta beyond 50% of its normal diameter, is a common and potentially life-threatening vascular disease. The molecular and cellular mechanisms underlying AAA pathogenesis remain unclear. Healthy endothelial cells (ECs) play a critical role in maintaining vascular homeostasis by regulating vascular tone and maintaining an anti-inflammatory, anti-thrombotic local environment. Increasing evidence indicates that endothelial dysfunction is an early pathologic event in AAA formation, contributing to both oxidative stress and inflammation in the degenerating arterial wall. Recent studies utilizing single-cell RNA sequencing revealed heterogeneous EC sub-populations, as determined by their transcriptional profiles, in aortic aneurysm tissue. This review summarizes recent findings, including clinical evidence of endothelial dysfunction in AAA, the impact of biomechanical stress on EC in AAA, the role of endothelial nitric oxide synthase (eNOS) uncoupling in AAA, and EC heterogeneity in AAA. These studies help to improve our understanding of AAA pathogenesis and ultimately may lead to the generation of EC-targeted therapeutics to treat or prevent this deadly disease.
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Aneurisma da Aorta Abdominal , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Dilatação Patológica , Células Endoteliais/patologia , Humanos , Estresse OxidativoRESUMO
Objective: Venous thromboembolism is a disease that encompasses both deep vein thrombosis and pulmonary embolism. Recent investigations have shown that receptor interacting protein kinase 3 (RIPK3), a protein known for its role in the programmed form of cell death necroptosis, may play a role in thrombosis. Specifically, RIPK3 has been shown to promote platelet activation in arterial thrombosis and mixed lineage kinase domain-like pseudokinase (MLKL), a protein downstream of RIPK3 in the necroptosis pathway, has been shown to promote neutrophil extracellular trap formation in deep vein thrombosis. This investigation sought to comprehensively investigate the role of RIPK3 in deep vein thrombogenesis. Methods: The inferior vena cava ligation and stenosis models of deep vein thrombosis were used in C57BL/6J, RIPK3 wild-type (Ripk3 +/+ ) and RIPK3-deficient (Ripk3 -/- ) mice. Downstream tissue analyses included measurement of thrombus weight and histological and Western blot analysis of tissues for markers of necroptosis and cell death. A subset of C57BL/6J mice were treated with a RIPK3 inhibitor to determine the effect on venous thrombosis. Results: C57BL/6J mice showed significant increases in thrombus weight from 6 to 48 hours. During the same time frame, RIPK3 progressively accumulated in the vein wall (a 35-fold increase from 0 to 48 hours). RIPK3 was present in the thrombus; however, it decreased with time. Although present in the thrombus, MLKL was nearly undetectable in the vein wall by Western blot at any timepoint. Immunostaining confirmed the high accumulation of RIPK3 in the vein wall, primarily colocalized to endothelial and smooth muscle cells. Phosphorylated MLKL, the active form of MLKL and executioner of necroptotic cell death, was detectable by immunostaining in the thrombus, but was present at low to undetectable levels in the vein wall. Propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed a high burden of necrotic and apoptotic cells within the thrombus at 48 hours, but a relatively lower burden within the vein wall. Despite robust accumulation of RIPK3 within the vessel wall and the thrombus, knockout and inhibition of RIPK3 failed to impact thrombus incident or weight at 48 hours after inferior vena cava ligation. Neutrophil extracellular trap burden did not differ between Ripk3 +/+ and Ripk3 -/- mice. Conclusions: In mice, the vein wall responded to deep vein thrombosis induction with elevation of RIPK3 without showing markers of necroptosis and apoptosis. Studies using genetic or pharmacological inhibition of RIPK3 suggest that this cell death mediator may not have a major role in the acute phase of venous thrombogenesis. Further investigation is needed to determine if RIPK3 plays a potentially non-necroptotic role within the vein wall during later stages of thrombus resolution and vein wall remodeling.
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Receptor interacting protein kinase 3 (RIPK3)-mediated smooth muscle cell (SMC) necroptosis has been shown to contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). However, the signaling steps downstream from RIPK3 during SMC necroptosis remain unknown. In this study, the roles of mixed lineage kinase domain-like pseudokinase (MLKL) and calcium/calmodulin-dependent protein kinase II (CaMKII) in SMC necroptosis were investigated. We found that both MLKL and CaMKII were phosphorylated in SMCs in a murine CaCl2-driven model of AAA and that Ripk3 deficiency reduced the phosphorylation of MLKL and CaMKII. In vitro, mouse aortic SMCs were treated with tumor necrosis factor α (TNFα) plus Z-VAD-FMK (zVAD) to induce necroptosis. Our data showed that both MLKL and CaMKII were phosphorylated after TNFα plus zVAD treatment in a time-dependent manner. SiRNA silencing of Mlkl-diminished cell death and administration of the CaMKII inhibitor myristoylated autocamtide-2-related inhibitory peptide (Myr-AIP) or siRNAs against Camk2d partially inhibited necroptosis. Moreover, knocking down Mlkl decreased CaMKII phosphorylation, but silencing Camk2d did not affect phosphorylation, oligomerization, or trafficking of MLKL. Together, our results indicate that both MLKL and CaMKII are involved in RIPK3-mediated SMC necroptosis, and that MLKL is likely upstream of CaMKII in this process.
Assuntos
Aneurisma da Aorta Abdominal/patologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos de Músculo Liso/patologia , Necrose , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Cloreto de Cálcio/toxicidade , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: Platelets are known to play an important role in venous thrombogenesis, but their role in thrombus maturation, resolution, and postthrombotic vein wall remodeling is unclear. The purpose of this study was to determine the role that circulating platelets play in the later phases of venous thrombosis. METHODS: We used a murine inferior vena cava (IVC) stenosis model. Baseline studies in untreated mice were performed to determine an optimal postthrombotic time point for tissue harvest that would capture both thrombus maturation/resolution and postthrombotic vein wall remodeling. This time point was found to be postoperative day 10. After undergoing IVC ultrasound on day 2 to confirm venous thrombus formation, mice were treated with a daily injection of platelet-depleting antibody (anti-GP1bα) to maintain thrombocytopenia or with control IgG until postoperative day 10, at which time IVC and thrombi were harvested and thrombus length, volume, fibrosis, neovascularization, and smooth muscle cell invasion analyzed. Vein wall fibrosis and intimal thickening were also determined. RESULTS: Mice that were made thrombocytopenic after venous thrombogenesis had thrombi that were less fibrotic, with fewer invading smooth muscle cells. Furthermore, thrombocytopenia in the setting of venous thrombosis resulted in less postthrombotic vein wall intimal thickening. Thrombus volume did not differ between thrombocytopenic mice and their control peers. CONCLUSIONS: This work suggests that circulating platelets contribute to venous thrombus maturation, fibrosis, and adverse vein wall remodeling, and that that inhibition of platelet recruitment may decrease thrombus and vein wall fibrosis, thus helping thrombolysis and preventing postthrombotic syndrome.