RESUMO
Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.
RESUMO
Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.
Assuntos
Hipertensão Pulmonar/patologia , MAP Quinase Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinase 3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Remodelação Vascular/fisiologia , Animais , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipertensão Pulmonar/metabolismo , Isquemia , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 3/genética , Camundongos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Moduladores Seletivos de Receptor Estrogênico/toxicidade , Transdução de Sinais , Tamoxifeno/toxicidade , Fator de Crescimento Transformador beta/genéticaRESUMO
Despite the clinical importance of arteriogenesis, this biological process is poorly understood. ERK1 and ERK2 are key components of a major intracellular signaling pathway activated by vascular endothelial growth (VEGF) and FGF2, growth factors critical to arteriogenesis. To investigate the specific role of each ERK isoform in arteriogenesis, we used mice with a global Erk1 knockout as well as Erk1 and Erk2 floxed mice to delete Erk1 or Erk2 in endothelial cells, macrophages, and smooth muscle cells. We found that ERK1 controls macrophage infiltration following an ischemic event. Loss of ERK1 in endothelial cells and macrophages induced an excessive macrophage infiltration leading to an increased but poorly functional arteriogenesis. Loss of ERK2 in endothelial cells leads to a decreased arteriogenesis due to decreased endothelial cell proliferation and a reduced eNOS expression. These findings show for the first time that isoform-specific roles of ERK1 and ERK2 in the control of arteriogenesis.
Assuntos
Artérias/enzimologia , Artérias/crescimento & desenvolvimento , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. METHODS: AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification. RESULTS: As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001). CONCLUSIONS: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.
Assuntos
Dependovirus/genética , Regulação da Expressão Gênica/fisiologia , Músculo Esquelético/metabolismo , Neuraminidase/metabolismo , Doença Arterial Periférica/metabolismo , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Simportadores/farmacocinética , Animais , Modelos Animais de Doenças , Membro Posterior/irrigação sanguínea , Isquemia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/diagnóstico por imagem , Solução Salina/administração & dosagemRESUMO
The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.
Assuntos
Neovascularização Fisiológica/genética , Sindecana-2/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Camundongos , Domínios Proteicos , Retina/crescimento & desenvolvimento , Análise de Sequência de Proteína , Sindecana-2/genética , Sindecana-2/metabolismo , Sindecana-4/genética , Sindecana-4/metabolismo , Sindecana-4/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Regulation of VEGFR2 represents an important mechanism for the control of angiogenesis. VEGFR2 activity can be regulated by post-translational modifications such as ubiquitination and acetylation. However, whether VEGFR2 can be regulated by SUMOylation has not been investigated. Here we show that endothelial-specific deletion of the SUMO endopeptidase SENP1 reduces pathological angiogenesis and tissue repair during hindlimb ischemia, and VEGF-induced angiogenesis in the cornea, retina, and ear. SENP1-deficient endothelial cells show increased SUMOylation of VEGFR2 and impaired VEGFR2 signalling. SUMOylation at lysine 1270 retains VEGFR2 in the Golgi and reduces its surface expression, attenuating VEGFR2-dependent signalling. Moreover, we find that SENP1 is downregulated and VEGFR2 hyper-SUMOylated in diabetic settings and that expression of a non-SUMOylated form of VEGFR2 rescues angiogenic defects in diabetic mice. These results show that VEGFR2 is regulated by deSUMOylation during pathological angiogenesis, and propose SENP1 as a potential therapeutic target for the treatment of diabetes-associated angiogenesis.
Assuntos
Espaço Intracelular/metabolismo , Neovascularização Patológica/metabolismo , Sumoilação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Córnea/irrigação sanguínea , Cisteína Endopeptidases/metabolismo , Diabetes Mellitus/patologia , Endopeptidases/metabolismo , Deleção de Genes , Técnicas de Introdução de Genes , Inativação Gênica , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , Retina/patologia , Proteína SUMO-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/químicaRESUMO
BACKGROUND: Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti-inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model. METHODS AND RESULTS: α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF-κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation. CONCLUSIONS: Blocking the fibronectin-Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.
Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Integrina alfa2/metabolismo , Integrina alfa5/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Placa Aterosclerótica , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrose , Predisposição Genética para Doença , Membro Posterior , Mediadores da Inflamação/metabolismo , Integrina alfa2/genética , Integrina alfa5/genética , Isquemia/genética , Isquemia/fisiopatologia , Leucócitos/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Fenótipo , Transdução de Sinais , Remodelação VascularRESUMO
Hyperoxia-induced acute lung injury (HALI) is a key contributor to the pathogenesis of bronchopulmonary dysplasia (BPD) in neonates, for which no specific preventive or therapeutic agent is available. Here we show that lung micro-RNA (miR)-34a levels are significantly increased in lungs of neonatal mice exposed to hyperoxia. Deletion or inhibition of miR-34a improves the pulmonary phenotype and BPD-associated pulmonary arterial hypertension (PAH) in BPD mouse models, which, conversely, is worsened by miR-34a overexpression. Administration of angiopoietin-1, which is one of the downstream targets of miR34a, is able to ameliorate the BPD pulmonary and PAH phenotypes. Using three independent cohorts of human samples, we show that miR-34a expression is increased in type 2 alveolar epithelial cells in neonates with respiratory distress syndrome and BPD. Our data suggest that pharmacologic miR-34a inhibition may be a therapeutic option to prevent or ameliorate HALI/BPD in neonates.
Assuntos
Angiopoietina-1/metabolismo , Displasia Broncopulmonar/metabolismo , Hiperóxia , Pulmão/metabolismo , MicroRNAs/genética , Receptor TIE-2/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/patologia , Biologia Computacional , Feminino , Deleção de Genes , Humanos , Recém-Nascido , Pulmão/patologia , Masculino , MicroRNAs/metabolismo , Fenótipo , Transdução de SinaisRESUMO
The surgical repair of heart and vascular disease often requires implanting synthetic grafts. While synthetic grafts have been successfully used for medium-to-large sized arteries, applications for small diameter arteries (<6 mm) is limited due to high rates of occlusion by thrombosis. Our objective was to develop a tissue engineered vascular graft (TEVG) for small diameter arteries. TEVGs composed of polylactic acid nanofibers with inner luminal diameter between 0.5 and 0.6 mm were surgically implanted as infra-renal aortic interposition conduits in 25 female C17SCID/bg mice. Twelve mice were given sham operations. Survival of mice with TEVG grafts was 91.6% at 12 months post-implantation (sham group: 83.3%). No instances of graft stenosis or aneurysmal dilatation were observed over 12 months post-implantation, assessed by Doppler ultrasound and microCT. Histologic analysis of explanted TEVG grafts showed presence of CD31-positive endothelial monolayer and F4/80-positive macrophages after 4, 8, and 12 months in vivo. Cells positive for α-smooth muscle actin were observed within TEVG, demonstrating presence of smooth muscle cells (SMCs). Neo-extracellular matrix consisting mostly of collagen types I and III were observed at 12 months post-implantation. PCR analysis supports histological observations. TEVG group showed significant increases in expressions of SMC marker, collagen-I and III, matrix metalloproteinases-2 and 9, and itgam (a macrophage marker), when compared to sham group. Overall, patency rates were excellent at 12 months after implantation, as structural integrity of these TEVG. Tissue analysis also demonstrated vessel remodeling by autologous cell.
Assuntos
Prótese Vascular , Nanofibras/química , Nanotecnologia/métodos , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Prótese Vascular/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Feminino , Ácido Láctico/efeitos adversos , Ácido Láctico/química , Ácido Láctico/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Nanofibras/efeitos adversos , Neovascularização Fisiológica/efeitos dos fármacos , Poliésteres , Polímeros/efeitos adversos , Polímeros/química , Polímeros/farmacologia , Análise de Sobrevida , Fatores de Tempo , Alicerces Teciduais/químicaRESUMO
Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), which encodes an eukaryotic translation initiation factor 4E transporter (4E-T) protein and suppresses the expression of capped mRNAs. In the absence of ELAVL1, skipping of exon 11 of Eif4enif1 forms the stable, short isoform, 4E-Ts. This alternative splicing event results in the formation of RNA processing bodies (PBs), enhanced turnover of angiogenic mRNAs, and suppressed sprouting behavior of vascular endothelial cells. Further, endothelial-specific Elavl1 knockout mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in oncogene-induced mammary cancer, resulting in attenuated blood flow and tumor growth, respectively. ELAVL1-regulated alternative splicing of Eif4enif1 leading to enhanced formation of PB and mRNA turnover constitutes a novel posttranscriptional mechanism critical for pathological angiogenesis.
Assuntos
Processamento Alternativo/fisiologia , Proteínas ELAV/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Proteína Semelhante a ELAV 1 , Éxons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismoRESUMO
Myeloid cells are important contributors to arteriogenesis, but their key molecular triggers and cellular effectors are largely unknown. We report, in inflammatory monocytes, that the combination of chemokine receptor (CCR2) and adhesion receptor (ß2 integrin) engagement leads to an interaction between activated Rac2 and Myosin 9 (Myh9), the heavy chain of Myosin IIA, resulting in augmented vascular endothelial growth factor A (VEGF-A) expression and induction of arteriogenesis. In human monocytes, CCL2 stimulation coupled to ICAM-1 adhesion led to rapid nuclear-to-cytosolic translocation of the RNA-binding protein HuR. This activation of HuR and its stabilization of VEGF-A mRNA were Rac2-dependent, and proteomic analysis for Rac2 interactors identified the 226 kD protein Myh9. The level of induced Rac2-Myh9 interaction strongly correlated with the degree of HuR translocation. CCL2-coupled ICAM-1 adhesion-driven HuR translocation and consequent VEGF-A mRNA stabilization were absent in Myh9(-/-) macrophages. Macrophage VEGF-A production, ischemic tissue VEGF-A levels, and flow recovery to hind limb ischemia were impaired in myeloid-specific Myh9(-/-) mice, despite preserved macrophage recruitment to the ischemic muscle. Micro-CT arteriography determined the impairment to be defective induced arteriogenesis, whereas developmental vasculogenesis was unaffected. These results place the macrophage at the center of ischemia-induced arteriogenesis, and they establish a novel role for Myosin IIA in signal transduction events modulating VEGF-A expression in tissue.
Assuntos
Antígenos CD18/metabolismo , Neovascularização Fisiológica/fisiologia , Miosina não Muscular Tipo IIA/metabolismo , Estabilidade de RNA/fisiologia , Receptores CCR2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Artérias/crescimento & desenvolvimento , Primers do DNA/genética , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Estabilidade de RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/genética , Microtomografia por Raio-X , Proteína RAC2 de Ligação ao GTPRESUMO
BACKGROUND: Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis. METHODS AND RESULTS: We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis, and arteriogenesis in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of extracellular signal-regulated kinase signaling, sprouting, migration, and proliferation compared with controls. Endothelial PTP1b null mice had increased retinal and Matrigel implant angiogenesis and accelerated wound healing, pointing to enhanced angiogenesis. Increased arteriogenesis was demonstrated by observations of faster recovery of arterial blood flow and large numbers of newly formed arterioles in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice. CONCLUSIONS: PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation.
Assuntos
Células Endoteliais/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta/citologia , Movimento Celular/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Feminino , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Neovascularização Fisiológica/fisiologia , Cultura Primária de Células , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , RNA Interferente Pequeno/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Transport distraction osteogenesis (DO) can be used to autologously reconstitute calvarial defects. The purpose of this study is to histomorphologically interrogate osteogenic formation during cranial transport distraction using a novel device. We also evaluate the effect of fat grafting on the regenerate and soft-tissue stability during distraction. This study was approved by Yale IACUC. Ten male New Zealand white rabbits (3 mo; 3.5 kg) were used (8 treatment, 2 control). A 16 × 16 mm defect was created abutted by a 10 × 16 mm transport disc. The device was fixated anterioposteriorly. Four animals were fat-grafted using 2 mL of subdermal intrascapular fat deposited along the distraction site. Latency (1 d), active distraction (12-14 d) (1.5 mm/d), and consolidation (4 wk) followed. Calcein and xylene orange fluorochromes were injected subcutaneously during and post-distraction to mark sites of bone formation. Following sacrifice, osteogenesis was assessed using microCT, histology, and fluorescence. Treatment animals demonstrated regenerate bone between distracted segments on microCT. MicroCT analysis of non-fat-grafted and fat-grafted animals revealed a mean density of 2271.95 mgHA/ccm and 2254.27 mgHA/ccm (P = 0.967), respectively, and defect bone versus total volume (BV/TV) of 0.0999 and 0.0766 (P = 0.5979), respectively. Controls had minimal reossification. Histologically, mean densities measured 43.63% and 8.19%, respectively. Fluorescence revealed ossification from the callus as well as from dura and periosteum in the cranial defect. Transport distraction is effective to reconstruct critically sized rabbit calvarial defects. Regenerate bone arises predominantly from the callus with contribution from surrounding dura and periosteum. Adipose grafting is well tolerated but does not enhance osseous regeneration.
Assuntos
Tecido Adiposo/transplante , Osteogênese por Distração/métodos , Procedimentos de Cirurgia Plástica/métodos , Crânio/cirurgia , Animais , Densidade Óssea/fisiologia , Doenças Ósseas/cirurgia , Regeneração Óssea/fisiologia , Calo Ósseo/patologia , Dura-Máter/patologia , Fluoresceínas , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador/métodos , Masculino , Osteogênese/fisiologia , Osteogênese por Distração/instrumentação , Periósteo/patologia , Coelhos , Distribuição Aleatória , Procedimentos de Cirurgia Plástica/instrumentação , Microtomografia por Raio-X/métodosRESUMO
Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.
Assuntos
Isquemia Miocárdica/prevenção & controle , Miocárdio/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Adenoviridae/genética , Animais , Vetores Genéticos/metabolismo , Coração/diagnóstico por imagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Ratos Wistar , Transdução de Sinais , Tomografia Computadorizada por Raios X , Regulação para Cima , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genéticaRESUMO
Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.
Assuntos
Angiopoietina-2/metabolismo , Células Endoteliais/metabolismo , Exossomos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Sindecana-4/metabolismo , Sinteninas/metabolismo , Angiopoietina-2/genética , Animais , Células Cultivadas , Células Endoteliais/citologia , Exossomos/genética , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Sindecana-4/genética , Sinteninas/genéticaRESUMO
Myocardial hypertrophy is an adaptation to increased hemodynamic demands. An increase in heart tissue must be matched by a corresponding expansion of the coronary vasculature to maintain and adequate supply of oxygen and nutrients for the heart. The physiological mechanisms that underlie the coordination of angiogenesis and cardiomyocyte growth are unknown. We report that induction of myocardial angiogenesis promotes cardiomyocyte growth and cardiac hypertrophy through a novel NO-dependent mechanism. We used transgenic, conditional overexpression of placental growth factor (PlGF) in murine cardiac tissues to stimulate myocardial angiogenesis and increase endothelial-derived NO release. NO production, in turn, induced myocardial hypertrophy by promoting proteasomal degradation of regulator of G protein signaling type 4 (RGS4), thus relieving the repression of the Gßγ/PI3Kγ/AKT/mTORC1 pathway that stimulates cardiomyocyte growth. This hypertrophic response was prevented by concomitant transgenic expression of RGS4 in cardiomyocytes. NOS inhibitor L-NAME also significantly attenuated RGS4 degradation, and reduced activation of AKT/mTORC1 signaling and induction of myocardial hypertrophy in PlGF transgenic mice, while conditional cardiac-specific PlGF expression in eNOS knockout mice did not induce myocardial hypertrophy. These findings describe a novel NO/RGS4/Gßγ/PI3Kγ/AKT mechanism that couples cardiac vessel growth with myocyte growth and heart size.
Assuntos
Crescimento Celular , Miócitos Cardíacos/fisiologia , Neovascularização Fisiológica , Óxido Nítrico/fisiologia , Proteínas RGS/metabolismo , Adaptação Biológica , Animais , Células Cultivadas , Vasos Coronários/fisiologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos , Miócitos Cardíacos/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Fator de Crescimento Placentário , Proteínas da Gravidez/fisiologia , Proteínas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TORRESUMO
Arterial morphogenesis is one of the most critical events during embryonic vascular development. Although arterial fate specification is mainly controlled by the Notch signaling pathway, arterial-venous patterning is modulated by a number of guidance factors. How these pathways are regulated is still largely unknown. Here, we demonstrate that endothelial activation of RAF1/extracellular signal-regulated kinase (ERK) pathway regulates arterial morphogenesis and arterial-venous patterning via Δ/Notch and semaphorin signaling. Introduction of a single amino acid RAF1 mutant (RAF1 Ser259Ala), which renders it resistant to inhibition by phosphorylation, into endothelial cells in vitro induced expression of virtually the entire embryonic arteriogenic program and activated semaphorin 6A-dependent endothelial cell-cell repulsion. In vivo, endothelial-specific expression of RAF1(S259A) during development induced extensive arterial morphogenesis both in the yolk sac and the embryo proper and disrupted arterial-venous patterning. Our results suggest that endothelial ERK signaling is critical for both arteriogenesis and arterial-venous patterning and that RAF1 Ser(259) phosphorylation plays a critical role in preventing unopposed ERK activation.
Assuntos
Artérias/embriologia , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Morfogênese , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Artérias/metabolismo , Células Cultivadas , Embrião de Mamíferos , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Gravidez , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/fisiologia , Semaforinas/genéticaRESUMO
BACKGROUND: Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. METHODS AND RESULTS: We used an inhibitor of NFκB activation (IκBαSR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-κB activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-κB activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1α levels, and a marked decrease in δ-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. CONCLUSIONS: These findings identify nuclear factor-κB as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-κB achieves this by regulating hypoxia-inducible factor-1α-dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating δ-like ligand 4 expression, which in turn determines the size and complexity of the network.
Assuntos
Células Endoteliais/metabolismo , Isquemia/fisiopatologia , Subunidade p50 de NF-kappa B/metabolismo , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Animais , Animais Recém-Nascidos , Becaplermina , Encéfalo/metabolismo , Modelos Animais de Doenças , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Camundongos , Camundongos Transgênicos , Subunidade p50 de NF-kappa B/genética , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor (VEGF)-A regulates angiogenesis, vascular morphology and permeability by signaling through its receptor VEGFR-2. The Shb adapter protein has previously been found to relay certain VEGFR-2 dependent signals and consequently vascular physiology and structure was assessed in Shb knockout mice. X-ray computed tomography of vessels larger than 24 µm diameter (micro-CT) after contrast injection revealed an increased frequency of 48-96 µm arterioles in the hindlimb calf muscle in Shb knockout mice. Intravital microscopy of the cremaster muscle demonstrated a less regular vasculature with fewer branch points and increased vessel tortuosity, changes that led to an increased blood flow velocity. Reduced in vivo angiogenesis was observed in Shb knockout Matrigel™ plugs. Unlike the wild-type situation, VEGF-A did not provoke a dissociation of VE-cadherin from adherens junctions in Shb knockout venules. The reduced angiogenesis and altered properties of junctions had consequences for two patho-physiological responses to arterial occlusion: vascular permeability was reduced in the Shb knockout cremaster muscle after ligation of one supplying artery and heat-induced blood flow determined by Laser-Doppler measurements was decreased in the hindlimb after ligation of the femoral artery. Consequently, the Shb knockout mouse exhibited structural and functional (angiogenesis and vascular permeability) vascular abnormalities that have implications for understanding the function of VEGF-A under physiological conditions.