RESUMEN
BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.
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Células Progenitoras Endoteliales , Hemangioma Cavernoso del Sistema Nervioso Central , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal , Animales , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Ratones , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/patología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/irrigación sanguínea , Ratones Noqueados , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Ratones Endogámicos C57BL , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genéticaRESUMEN
BACKGROUND: Angiogenesis, which plays a critical role in embryonic development and tissue repair, is controlled by a set of angiogenic signaling pathways. As a TF (transcription factor) belonging to the basic helix-loop-helix family, HEY (hairy/enhancer of split related with YRPW motif)-1 (YRPW motif, abbreviation of 4 highly conserved amino acids in the motif) has been identified as a key player in developmental angiogenesis. However, the precise mechanisms underlying HEY1's actions in angiogenesis remain largely unknown. Our previous studies have suggested a potential role for posttranslational SUMOylation in the dynamic regulation of vascular development and organization. METHODS: Immunoprecipitation, mass spectrometry, and bioinformatics analysis were used to determine the biochemical characteristics of HEY1 SUMOylation. The promoter-binding capability of HEY1 was determined by chromatin immunoprecipitation, dual luciferase, and electrophoretic mobility shift assays. The dimerization pattern of HEY1 was determined by coimmunoprecipitation. The angiogenic capabilities of endothelial cells were assessed by CCK-8 (cell counting kit-8), 5-ethynyl-2-deoxyuridine staining, wound healing, transwell, and sprouting assays. Embryonic and postnatal vascular growth in mouse tissues, matrigel plug assay, cutaneous wound healing model, oxygen-induced retinopathy model, and tumor angiogenesis model were used to investigate the angiogenesis in vivo. RESULTS: We identified intrinsic endothelial HEY1 SUMOylation at conserved lysines by TRIM28 (tripartite motif containing 28) as the unique E3 ligase. Functionally, SUMOylation facilitated HEY1-mediated suppression of angiogenic RTK (receptor tyrosine kinase) signaling and angiogenesis in primary human endothelial cells and mice with endothelial cell-specific expression of wild-type HEY1 or a SUMOylation-deficient HEY1 mutant. Mechanistically, SUMOylation facilitates HEY1 homodimer formation, which in turn preserves HEY1's DNA-binding capability via recognition of E-box promoter elements. Therefore, SUMOylation maintains HEY1's function as a repressive TF controlling numerous angiogenic genes, including RTKs and Notch pathway components. Proangiogenic stimuli induce HEY1 deSUMOylation, leading to heterodimerization of HEY1 with HES (hairy and enhancer of split)-1, which results in ineffective DNA binding and loss of HEY1's angiogenesis-suppressive activity. CONCLUSIONS: Our findings demonstrate that reversible HEY1 SUMOylation is a molecular mechanism that coordinates endothelial angiogenic signaling and angiogenesis, both in physiological and pathological milieus, by fine-tuning the transcriptional activity of HEY1. Specifically, SUMOylation facilitates the formation of the HEY1 transcriptional complex and enhances its DNA-binding capability in endothelial cells.
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Células Endoteliales , Sumoilación , Animales , Humanos , Ratones , Angiogénesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Células Endoteliales/metabolismoRESUMEN
BACKGROUND: Hypoxia is a major cause and promoter of pulmonary hypertension (PH), a representative vascular remodeling disease with poor prognosis and high mortality. However, the mechanism underlying how pulmonary arterial system responds to hypoxic stress during PH remains unclear. Endothelial mitochondria are considered signaling organelles on oxygen tension. Results from previous clinical research and our studies suggested a potential role of posttranslational SUMOylation (small ubiquitin-like modifier modification) in endothelial mitochondria in hypoxia-related vasculopathy. METHODS: Chronic hypoxia mouse model and Sugen/hypoxia rat model were employed as PH animal models. Mitochondrial morphology and subcellular structure were determined by transmission electron and immunofluorescent microscopies. Mitochondrial metabolism was determined by mitochondrial oxygen consumption rate and extracellular acidification rate. SUMOylation and protein interaction were determined by immunoprecipitation. RESULTS: The involvement of SENP1 (sentrin-specific protease 1)-mediated SUMOylation in mitochondrial remodeling in the pulmonary endothelium was identified in clinical specimens of hypoxia-related PH and was verified in human pulmonary artery endothelial cells under hypoxia. Further analyses in clinical specimens, hypoxic rat and mouse PH models, and human pulmonary artery endothelial cells and human embryonic stem cell-derived endothelial cells revealed that short-term hypoxia-induced SENP1 translocation to endothelial mitochondria to regulate deSUMOylation (the reversible process of SUMOylation) of mitochondrial fission protein FIS1 (mitochondrial fission 1), which facilitated FIS1 assembling with fusion protein MFN2 (mitofusin 2) and mitochondrial gatekeeper VDAC1 (voltage-dependent anion channel 1), and the membrane tethering activity of MFN2 by enhancing its oligomerization. Consequently, FIS1 deSUMOylation maintained the mitochondrial integrity and endoplasmic reticulum-mitochondria calcium communication across mitochondrial-associated membranes, subsequently preserving pulmonary endothelial function and vascular homeostasis. In contrast, prolonged hypoxia disabled the FIS1 deSUMOylation by diminishing the availability of SENP1 in mitochondria via inducing miR (micro RNA)-138 and consequently resulted in mitochondrial dysfunction and metabolic reprogramming in pulmonary endothelium. Functionally, introduction of viral-packaged deSUMOylated FIS1 within pulmonary endothelium in mice improved pulmonary endothelial dysfunction and hypoxic PH development, while knock-in of SUMO (small ubiquitin-like modifier)-conjugated FIS1 in mice exaggerated the diseased cellular and tissue phenotypes. CONCLUSIONS: By maintaining endothelial mitochondrial homeostasis, deSUMOylation of FIS1 adaptively preserves pulmonary endothelial function against hypoxic stress and consequently protects against PH. The FIS1 deSUMOylation-SUMOylation transition in pulmonary endothelium is an intrinsic pathogenesis of hypoxic PH.
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Hipertensión Pulmonar , Enfermedades Vasculares , Humanos , Ratones , Ratas , Animales , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/prevención & control , Células Endoteliales , Mitocondrias , Modelos Animales de Enfermedad , Endotelio , Ubiquitinas , Proteínas de la Membrana , Proteínas MitocondrialesRESUMEN
Angiogenesis contributes fundamentally to embryonic development, tissue homeostasis, and wound healing. Basic fibroblast growth factor (FGF2) is recognized as the first proangiogenic molecule discovered, and it facilitates angiogenesis by activating FGF receptor 1 (FGFR1) signaling in endothelial cells. However, the precise roles of FGFR and the FGF/FGFR signaling axis in angiogenesis remain unclear, especially because of the contradictory phenotypes of in vivo FGF and FGFR gene deficiency models. Our previous study results suggested a potential role of posttranslational small ubiquitin-like modifier modification (SUMOylation), with highly dynamic regulatory features, in vascular development and disorder. Here, we identified SENP1-regulated endothelial FGFR1 SUMOylation at conserved lysines responding to proangiogenic stimuli, while SENP1 functioned as the deSUMOylase. Hypoxia-enhanced FGFR1 SUMOylation restricted the tyrosine kinase activation of FGFR1 by modulating the dimerization of FGFR1 and FGFR1 binding with its phosphatase PTPRG. Consequently, it facilitated the recruitment of FRS2α to VEGFR2 but limited additional recruitment of FRS2α to FGFR1, supporting the activation of VEGFA/VEGFR2 signaling in endothelial cells. Furthermore, SUMOylation-defective mutation of FGFR1 resulted in exaggerated FGF2/FGFR1 signaling but suppressed VEGFA/VEGFR2 signaling and the angiogenic capabilities of endothelial cells, which were rescued by FRS2α overexpression. Reduced angiogenesis and endothelial sprouting in mice bearing an endothelial-specific, FGFR1 SUMOylation-defective mutant confirmed the functional significance of endothelial FGFR1 SUMOylation in vivo. Our findings identify the reversible SUMOylation of FGFR1 as an intrinsic fine-tuned mechanism in coordinating endothelial angiogenic signaling during neovascularization; SENP1-regulated FGFR1 SUMOylation and deSUMOylation controls the competitive recruitment of FRS2α by FGFR1 and VEGFR2 to switch receptor-complex formation responding to hypoxia and normoxia angiogenic environments.
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Células Endoteliales , Neovascularización Fisiológica , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Sumoilación , Animales , Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Hipoxia/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Sumoilación/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Six new iridoid glycosides were isolated from the ethyl acetate fraction of the whole plants of Hedyotis diffusa Willd. They were identified as E-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (1), Z-6-O-p-methoxycinnamoyl-10-O-acetyl scandoside acid methyl ester (2), E-6-O-caffeoyl scandoside methyl ester (3), E-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (4), Z-6-O-p-coumaroyl-6'-O-acetyl scandoside methyl ester (5), and E-6-O-p-coumaroyl-4'-O-acetyl scandoside methyl ester (6). The structures of them were elucidated based on unambiguous spectroscopic data (UV, IR, HRESIMS, and NMR). They were screened for anti-inflammatory effect, antioxidant effect, antitumor effect, and neuroprotective effect and did not show potent activities.
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Ácidos Cumáricos , Hedyotis , Glicósidos Iridoides , Glicósidos Iridoides/farmacología , Hedyotis/química , Antioxidantes , Espectroscopía de Resonancia Magnética , Ésteres , Glicósidos/farmacologíaRESUMEN
Hexarelin, a synthetic growth hormone-releasing peptide, is shown to be protective in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of hexarelin in abdominal aortic aneurysm (AAA) remains undefined. The present study determined the effect of hexarelin administration (200 µg/kg twice per day) in a mouse model of elastase-induced abdominal aortic aneurysm. Echocardiography and in situ pictures showed hexarelin decreased infrarenal aorta diameter. Histology staining showed elastin degradation was improved in hexarelin-treated group. Hexarelin rescued smooth muscle cell contractile phenotype with increased α-SMA and decreased MMP2. Furthermore, hexarelin inhibited inflammatory cell infiltration, NLRP3 inflammasome activation and IL-18 production. Particularly, hexarelin suppressed NF-κB signaling pathway which is a key initiator of inflammatory response. These results demonstrated that hexarelin attenuated AAA development by inhibiting SMC phenotype switch and NF-κB signaling mediated inflammatory response.
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Antiinflamatorios/farmacología , Aneurisma de la Aorta Abdominal/prevención & control , Plasticidad de la Célula/efectos de los fármacos , Inflamasomas/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Oligopéptidos/farmacología , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/inmunología , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamasomas/metabolismo , Masculino , Ratones Endogámicos C57BL , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fenotipo , Transducción de Señal , Remodelación Vascular/efectos de los fármacosRESUMEN
OBJECTIVE: Cerebral cavernous malformations (CCMs) can happen anywhere in the body, although they most commonly produce symptoms in the brain. The role of CCM genes in other vascular beds outside the brain and retina is not well-examined, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in lymphatics. Approach and Results: Mice with an inducible pan-endothelial cell (EC) or lymphatic EC deletion of Ccm3 (Pdcd10ECKO or Pdcd10LECKO) exhibit dilated lymphatic capillaries and collecting vessels with abnormal valve structure. Morphological alterations were correlated with lymphatic dysfunction in Pdcd10LECKO mice as determined by Evans blue dye and fluorescein isothiocyanate(FITC)-dextran transport assays. Pdcd10LECKO lymphatics had increased VEGFR3 (vascular endothelial growth factor receptor-3)-ERK1/2 (extracellular signal-regulated kinase 1/2) signaling with lymphatic hyperplasia. Mechanistic studies suggested that VEGFR3 is primarily regulated at a transcriptional level in Ccm3-deficient lymphatic ECs, in an NF-κB (nuclear factor κB)-dependent manner. CCM3 binds to importin alpha 2/KPNA2 (karyopherin subunit alpha 2), and a CCM3 deletion releases KPNA2 to activate NF-κB P65 by facilitating its nuclear translocation and P65-dependent VEGFR3 transcription. Moreover, increased VEGFR3 in lymphatic EC preferentially activates ERK1/2 signaling, which is critical for lymphatic EC proliferation. Importantly, inhibition of VEGFR3 or ERK1/2 rescued the lymphatic defects in structure and function. CONCLUSIONS: Our data demonstrate that CCM3 deletion augments the VEGFR3-ERK1/2 signaling in lymphatic EC that drives lymphatic hyperplasia and malformation and warrant further investigation on the potential clinical relevance of lymphatic dysfunction in patients with CCM.
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Endotelio Linfático/fisiopatología , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Sistema de Señalización de MAP Quinasas/fisiología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Células Endoteliales/fisiología , Endotelio Linfático/patología , Femenino , Eliminación de Gen , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hiperplasia , Masculino , Ratones Endogámicos , Modelos Animales , FN-kappa B/genética , Translocación GenéticaRESUMEN
BACKGROUND: Ischemia reperfusion injury (IRI) predisposes to the formation of donor-specific antibodies, a factor contributing to chronic rejection and late allograft loss. METHODS: We describe a mechanism underlying the correlative association between IRI and donor-specific antibodies by using humanized models and patient specimens. RESULTS: IRI induces immunoglobulin M-dependent complement activation on endothelial cells that assembles an NLRP3 (NOD-like receptor pyrin domain-containing protein 3) inflammasome via a Rab5-ZFYVE21-NIK axis and upregulates ICOS-L (inducible costimulator ligand) and PD-L2 (programmed death ligand 2). Endothelial cell-derived interleukin-18 (IL-18) selectively expands a T-cell population (CD4+CD45RO+PD-1hiICOS+CCR2+CXCR5-) displaying features of recently described T peripheral helper cells. This population highly expressed IL-18R1 and promoted donor-specific antibodies in response to IL-18 in vivo. In patients with delayed graft function, a clinical manifestation of IRI, these cells were Ki-67+IL-18R1+ and could be expanded ex vivo in response to IL-18. CONCLUSIONS: IRI promotes elaboration of IL-18 from endothelial cells to selectively expand alloreactive IL-18R1+ T peripheral helper cells in allograft tissues to promote donor-specific antibody formation.
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Células Endoteliales de la Vena Umbilical Humana/inmunología , Inmunoglobulina M/inmunología , Interleucina-18/inmunología , Isoanticuerpos/inmunología , Trasplante de Órganos , Daño por Reperfusión/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Funcionamiento Retardado del Injerto/inmunología , Funcionamiento Retardado del Injerto/patología , Femenino , Regulación de la Expresión Génica/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamasomas/inmunología , Subunidad alfa del Receptor de Interleucina-18 , Ratones , Ratones SCID , Daño por Reperfusión/patología , Transducción de Señal/inmunología , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
While emerging evidence suggests the link between endothelial activation of TGF-ß signaling, induction of endothelial-to-mesenchymal transition (EndMT), and cardiovascular disease (CVD), the molecular underpinning of this connection remains enigmatic. Here, we report aberrant expression of H19 lncRNA and TET1 in endothelial cells (ECs) of human atherosclerotic coronary arteries. Using primary human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAoECs) we show that TNF-α, a known risk factor for endothelial dysfunction and CVD, induces H19 expression which in turn activates TGF-ß signaling and EndMT via a TET1-dependent epigenetic mechanism. We also show that H19 regulates TET1 expression at the posttranscriptional level. Further, we provide evidence that this H19/TET1-mediated regulation of TGF-ß signaling and EndMT occurs in mouse pulmonary microvascular ECs in vivo under hyperglycemic conditions. We propose that endothelial activation of the H19/TET1 axis may play an important role in EndMT and perhaps CVD.
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Transición Epitelial-Mesenquimal/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Largo no Codificante/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Vasos Coronarios/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Postranscripcional del ARN/fisiología , Transducción de Señal/fisiologíaRESUMEN
RATIONALE: Complement activation contributes to multiple immune-mediated pathologies. In late allograft failure, donor-specific antibody deposits complement membrane attack complexes (MAC) on graft endothelial cells (ECs), substantially increasing their immunogenicity without causing lysis. Internalized MAC stabilize NIK (NF-κB [nuclear factor kappa-light-chain-enhancer of activated B cells]-inducing kinase) protein on Rab5+MAC+ endosomes, activating noncanonical NF-κB signaling. However, the link to increased immunogenicity is unclear. OBJECTIVE: To identify mechanisms by which alloantibody and internalized MAC activate ECs to enhance their ability to increase T-cell responses. METHODS AND RESULTS: In human EC cultures, internalized MAC also causes NLRP3 (NOD-like receptor family pyrin domain containing 3) translocation from endoplasmic reticulum to Rab5+MAC+NIK+ endosomes followed by endosomal NIK-dependent inflammasome assembly. Cytosolic NIK, stabilized by LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells), does not trigger inflammasome assembly, and ATP-triggered inflammasome assembly does not require NIK. IFN-γ (interferon-γ) primes EC responsiveness to MAC by increasing NLRP3, pro-caspase 1, and gasdermin D expression. NIK-activated noncanonical NF-κB signaling induces pro-IL (interleukin)-1ß expression. Inflammasome processed pro-IL-1ß, and gasdermin D results in IL-1ß secretion that increases EC immunogenicity through IL-1 receptor signaling. Activation of human ECs lining human coronary artery grafts in immunodeficient mouse hosts by alloantibody and complement similarly depends on assembly of an NLRP3 inflammasome. Finally, in renal allograft biopsies showing chronic rejection, caspase-1 is activated in C4d+ ECs of interstitial microvessels, supporting the relevance of the cell culture findings. CONCLUSIONS: In response to antibody-mediated complement activation, IFN-γ-primed human ECs internalize MAC, triggering both endosomal-associated NIK-dependent NLRP3 inflammasome assembly and IL-1 synthesis, resulting in autocrine/paracrine IL-1ß-mediated increases in EC immunogenicity. Similar responses may underlie other complement-mediated pathologies.
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Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Interferón gamma/farmacología , Interleucina-1/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Adulto , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inflamasomas/metabolismo , MasculinoRESUMEN
RATIONALE: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell-cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. OBJECTIVE: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. METHODS AND RESULTS: Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)-deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. CONCLUSIONS: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.
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Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Receptores Notch/metabolismo , Sumoilación , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión , Proteínas de Unión al Calcio , Células Cultivadas , Cisteína Endopeptidasas , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Unión Proteica , Receptores Notch/química , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN, the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined. APPROACH AND RESULTS: We quantified determinants of luminal stenosis in thoracic aortas of Eln-/- mice incompletely rescued by human ELN. Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject. CONCLUSIONS: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency.
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Aorta Torácica/crecimiento & desarrollo , Enfermedades de la Aorta/fisiopatología , Elastina/metabolismo , Síndrome de Williams/fisiopatología , Adulto , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Proliferación Celular , Células Cultivadas , Colágeno/metabolismo , Constricción Patológica , Modelos Animales de Enfermedad , Elastina/deficiencia , Elastina/genética , Fibrosis , Predisposición Genética a la Enfermedad , Humanos , Hiperplasia , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Músculo Liso Vascular/crecimiento & desarrollo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Fenotipo , Factores de Tiempo , Rigidez Vascular , Vasoconstricción , Síndrome de Williams/genética , Síndrome de Williams/metabolismo , Síndrome de Williams/patologíaRESUMEN
OBJECTIVE: Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln-null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln-null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term. APPROACH AND RESULTS: Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-ß-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective. CONCLUSIONS: In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy.
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Enfermedades de la Aorta/tratamiento farmacológico , Colágeno/metabolismo , Elastina/deficiencia , Mecanotransducción Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Rigidez Vascular/efectos de los fármacos , Síndrome de Williams/tratamiento farmacológico , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/enzimología , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/fisiopatología , Proliferación Celular/efectos de los fármacos , Elastina/genética , Everolimus/farmacología , Quinasa 1 de Adhesión Focal/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Mesilato de Imatinib/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Fenotipo , Fosforilación , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Síndrome de Williams/enzimología , Síndrome de Williams/patología , Síndrome de Williams/fisiopatologíaRESUMEN
Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB-inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5(+)endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC(+) endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt(+)NIK(+) signalosome on Rab5(+) endosomes.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Endosomas/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab5/metabolismo , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Clatrina/metabolismo , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Hidrazonas/farmacología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones SCID , Biosíntesis de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 3 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
The development of effective pharmacological treatment of abdominal aortic aneurysm (AAA) potentially offers great benefit to patients with preaneurysmal aortic dilation by slowing the expansion of aneurysms and reducing the need for surgery. To date, therapeutic targets for slowing aortic dilation have had low efficacy. Thus, in this study, we aim to elucidate possible mechanisms driving aneurysm progression to identify potential targets for pharmacological intervention. We demonstrate that mechanistic target of rapamycin (mTOR) signaling is overactivated in aortic smooth muscle cells (SMCs), which contributes to murine AAA. Rapamycin, a typical mTOR pathway inhibitor, dramatically limits the expansion of the abdominal aorta following intraluminal elastase perfusion. Furthermore, reduction of aortic diameter is achieved by inhibition of the mTOR pathway, which preserves and/or restores the contractile phenotype of SMCs and downregulates macrophage infiltration, matrix metalloproteinase expression, and inflammatory cytokine production. Taken together, these results highlight the important role of the mTOR cascade in aneurysm progression and the potential application of rapamycin as a therapeutic candidate for AAA.NEW & NOTEWORTHY This study provides novel observations that mechanistic target of rapamycin (mTOR) signaling is overactivated in aortic smooth muscle cells and contributes to mouse abdominal aortic aneurysm (AAA) and that rapamycin protects against aneurysm development. Our data highlight the importance of preservation and/or restoration of the smooth muscle cell contractile phenotype and reduction of inflammation by mTOR inhibition in AAA.
Asunto(s)
Antiinflamatorios/farmacología , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Músculo Liso Vascular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Vasoconstricción/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/enzimología , Aorta/patología , Aorta/fisiopatología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/fisiopatología , Citocinas/metabolismo , Dilatación Patológica , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones Endogámicos C57BL , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Elastasa Pancreática , Fenotipo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factores de TiempoRESUMEN
RATIONALE: Transplantation, the most effective therapy for end-stage organ failure, is markedly limited by early-onset cardiovascular disease (CVD) and premature death of the host. The mechanistic basis of this increased CVD is not fully explained by known risk factors. OBJECTIVE: To investigate the role of alloimmune responses in promoting CVD of organ transplant recipients. METHODS AND RESULTS: We established an animal model of graft-exacerbated host CVD by combining murine models of atherosclerosis (apolipoprotein E-deficient recipients on standard diet) and of intra-abdominal graft rejection (heterotopic cardiac transplantation without immunosuppression). CVD was absent in normolipidemic hosts receiving allogeneic grafts and varied in severity among hyperlipidemic grafted hosts according to recipient-donor genetic disparities, most strikingly across an isolated major histocompatibility complex class II antigen barrier. Host disease manifested as increased atherosclerosis of the aorta that also involved the native coronary arteries and new findings of decreased cardiac contractility, ventricular dilatation, and diminished aortic compliance. Exacerbated CVD was accompanied by greater levels of circulating cytokines, especially interferon-γ and other Th1-type cytokines, and showed both systemic and intralesional activation of leukocytes, particularly T-helper cells. Serological neutralization of interferon-γ after allotransplantation prevented graft-related atherosclerosis, cardiomyopathy, and aortic stiffening in the host. CONCLUSIONS: Our study reveals that sustained activation of the immune system because of chronic allorecognition exacerbates the atherogenic diathesis of hyperlipidemia and results in de novo cardiovascular dysfunction in organ transplant recipients.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Rechazo de Injerto/complicaciones , Trasplante de Corazón/efectos adversos , Hiperlipidemias/complicaciones , Mediadores de Inflamación/sangre , Interferón gamma/sangre , Aloinjertos , Animales , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E , Aterosclerosis/sangre , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Cardiomiopatías/sangre , Cardiomiopatías/etiología , Cardiomiopatías/inmunología , Cardiomiopatías/prevención & control , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/fisiopatología , Enfermedades Cardiovasculares/prevención & control , Modelos Animales de Enfermedad , Femenino , Rechazo de Injerto/sangre , Rechazo de Injerto/inmunología , Hemodinámica , Antígenos de Histocompatibilidad Clase II/inmunología , Hiperlipidemias/sangre , Hiperlipidemias/genética , Mediadores de Inflamación/inmunología , Interferón gamma/inmunología , Activación de Linfocitos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Células TH1/inmunología , Células TH1/metabolismo , Disfunción Ventricular Izquierda/sangre , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/inmunología , Disfunción Ventricular Izquierda/prevención & control , Función Ventricular IzquierdaRESUMEN
Vascular endothelial growth factors (VEGFs) signal via their cognate receptor tyrosine kinases designated VEGFR1-3. We report that the docking protein fibroblast growth factor receptor substrate 2 (FRS2α) plays a critical role in cell signaling via these receptors. In vitro FRS2α regulates VEGF-A and VEGF-C-dependent activation of extracellular signal-regulated receptor kinase signaling and blood and lymphatic endothelial cells migration and proliferation. In vivo endothelial-specific deletion of FRS2α results in the profound impairment of postnatal vascular development and adult angiogenesis, lymphangiogenesis, and arteriogenesis. We conclude that FRS2α is a previously unidentified component of VEGF receptors signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Transducción de Señal/fisiología , Animales , Movimiento Celular/fisiología , Cartilla de ADN/genética , Células Endoteliales , Perfilación de la Expresión Génica , Vectores Genéticos , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Flujometría por Láser-Doppler , Lentivirus , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Microtomografía por Rayos XRESUMEN
Cardiac allograft vasculopathy is the major cause of late graft loss in heart transplant recipients. Histological studies of characteristic end-stage lesions reveal arterial changes consisting of a diffuse, confluent, and concentric intimal expansion containing graft-derived cells expressing smooth muscle markers, extracellular matrix, penetrating microvessels, and a host mononuclear cell infiltrate concentrated subjacent to an intact graft-derived luminal endothelial cell lining with little evidence of acute injury. This intimal expansion combined with inadequate compensatory outward remodeling produces severe generalized stenosis extending throughout the epicardial and intramyocardial arterial tree that causes ischemic graft failure. Cardiac allograft vasculopathy lesions affect ≥50% of transplant recipients and are both progressive and refractory to treatment, resulting in ≈5% graft loss per year through the first 10 years after transplant. Lesions typically stop at the suture line, implicating alloimmunity as the primary driver, but pathogenesis may be multifactorial. Here, we will discuss 6 potential contributors to lesion formation (1) conventional risk factors of atherosclerosis; (2) pre- or peritransplant injuries; (3) infection; (4) innate immunity; (5) T-cell-mediated immunity; and (6) B-cell-mediated immunity through production of donor-specific antibody. Finally, we will consider how these various mechanisms may interact with each other.
Asunto(s)
Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/patología , Vasos Coronarios/trasplante , Trasplante de Corazón/efectos adversos , Aloinjertos , Animales , Linfocitos B/inmunología , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/patología , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/inmunología , Vasos Coronarios/inmunología , Vasos Coronarios/metabolismo , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunidad Innata , Isoanticuerpos/sangre , Factores de Riesgo , Transducción de Señal , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Smooth muscle cells (SMCs) are remarkably plastic. Their reversible differentiation is required for growth and wound healing but also contributes to pathologies such as atherosclerosis and restenosis. Although key regulators of the SMC phenotype, including myocardin (MYOCD) and KLF4, have been identified, a unifying epigenetic mechanism that confers reversible SMC differentiation has not been reported. METHODS AND RESULTS: Using human SMCs, human arterial tissue, and mouse models, we report that SMC plasticity is governed by the DNA-modifying enzyme ten-eleven translocation-2 (TET2). TET2 and its product, 5-hydroxymethylcytosine (5-hmC), are enriched in contractile SMCs but reduced in dedifferentiated SMCs. TET2 knockdown inhibits expression of key procontractile genes, including MYOCD and SRF, with concomitant transcriptional upregulation of KLF4. TET2 knockdown prevents rapamycin-induced SMC differentiation, whereas TET2 overexpression is sufficient to induce a contractile phenotype. TET2 overexpression also induces SMC gene expression in fibroblasts. Chromatin immunoprecipitation demonstrates that TET2 coordinately regulates phenotypic modulation through opposing effects on chromatin accessibility at the promoters of procontractile versus dedifferentiation-associated genes. Notably, we find that TET2 binds and 5-hmC is enriched in CArG-rich regions of active SMC contractile promoters (MYOCD, SRF, and MYH11). Loss of TET2 and 5-hmC positively correlates with the degree of injury in murine models of vascular injury and human atherosclerotic disease. Importantly, localized TET2 knockdown exacerbates injury response, and local TET2 overexpression restores the 5-hmC epigenetic landscape and contractile gene expression and greatly attenuates intimal hyperplasia in vivo. CONCLUSIONS: We identify TET2 as a novel and necessary master epigenetic regulator of SMC differentiation.