RESUMEN
Angiogenesis is a key player in the pathogenesis of rheumatoid arthritis. Exocytosis from Weibel-Palade bodies is a prerequisite for angiopoietin-2 (Ang-2) to activate endothelial cells and initiate angiogenesis. Geniposide (GE) was previously reported to exert anti-angiogenic effects. The aim of this study was to shed light on whether and how GE regulates Ang-2 exocytosis. A rat model of adjuvant arthritis (AA) was established to evaluate the therapeutic effect of GE (60 and 120 mg/kg) especially in synovial angiogenesis. In addition, the Matrigel plug assay was used to detect the effect of GE (120 and 240 mg/kg) on angiogenesis in AA mice. In vitro, sphingosine-1-phosphate (S1P)-stimulated human umbilical vein endothelial cells (HUVECs) were used to investigate the effect and mechanism of GE on Ang-2 exocytosis. It was found that GE improved the symptoms of AA rats and inhibited angiogenesis in AA, which may be related to the down-regulation of S1P receptors 1, 3 (S1PR1, S1PR3), phospholipase Cß3 (PLCß3), inositol 1,4,5-trisphosphate receptor (IP3 R) and Ang-2 expression. The results of in vitro experiments showed that S1P induced rapid release of Ang-2 from HUVECs with multigranular exocytosis. Suppression of the S1P/S1PR1/3/PLCß3/Ca2+ signal axis by the S1PR1/3 inhibitor VPC23019 and the IP3 R inhibitor 2-APB blocked Ang-2 exocytosis, accompanied by diminished angiogenesis in vitro. GE dose-dependently weakened S1P/S1PR1/3/PLCß3/Ca2+ signal axis activation, Ang-2 exocytosis and angiogenesis in HUVECs (p < 0.05, p < 0.01). Overall, these findings revealed that angiogenesis inhibition of GE was partly attributed to the intervention of Ang-2 exocytosis through negatively modulating the S1P/S1PR1/3/PLCß3/Ca2+ signal axis, providing a novel strategy for rheumatoid arthritis anti-angiogenic therapy.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Iridoides , Ratas , Humanos , Ratones , Animales , Angiopoyetina 2/farmacología , Angiogénesis , Células Endoteliales de la Vena Umbilical Humana , Exocitosis , Angiopoyetina 1/metabolismoRESUMEN
Objective To explore the effect and mechanism of penehyclidine hydrochloride (PHCD) on vascular endothelial injury in septic rats. Methods Fifty male SD rats were randomly divided into control group, lipopolysaccharide (LPS) induced sepsis group (model group), low dose PHCD (0.3 mg/kg) group, medium dose PHCD (1.0 mg/kg) group and high dose PHCD (3.0 mg/kg) groups, ten mice for each group. Normal saline was injected into the tail vein of the control group, and 10 mg/kg lipopolysaccharide (LPS) was injected into the tail vein of the rats in other groups to prepare the sepsis rat models. After the models were successfully established, low, medium and high doses (0.3, 1.0, 3.0 mg/kg) of PHCD solution were injected into the tail vein of the rats of corresponding groups. Wet/dry mass ratio (W/D) of lung tissue of rats in each group was measured, and ELISA was used to assay interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 content and rat plasma angiopoietin 2 (Ang2) content in bronchoalveolar lavage fluid (BALF). HE staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to observe the expression of Ang2 in the right lung tissues. Western blot analysis was performed to detect Ang2 and vascular endothelial cadherin (VE-cadherin) protein in lung tissues. Results Compared with the control group, the W/D ratio of the lung tissues of rats in the model group and the contents of IL-1ß, IL-6 and TNF-α in BALF were significantly increased; the lung tissues showed obvious pathological damage, with up-regulation of Ang2 expression and down-regulation of VE-Cadherin expression. Compared with the model group, the W/D ratio of the lung tissues of rats in three PHCD treatment groups and the contents of IL-1ß, IL-6 and TNF-α in BALF were significantly reduced; the pathological damage of lung tissue was significantly reduced, with down-regulation of Ang2 expression and up-regulation of VE-cadherin expression. Conclusion PHCD can reduce LPS-induced lung inflammation in rats with sepsis by regulating the Ang2/VE-Cadherin pathway, thereby improving vascular endothelial injury.
Asunto(s)
Lesión Pulmonar Aguda , Sepsis , Ratas , Ratones , Animales , Masculino , Lipopolisacáridos/toxicidad , Lipopolisacáridos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Angiopoyetina 2/metabolismo , Angiopoyetina 2/farmacología , Interleucina-6/metabolismo , Ratas Sprague-Dawley , Pulmón , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Sepsis/inducido químicamente , Sepsis/tratamiento farmacológico , Sepsis/metabolismoRESUMEN
Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), has been found to stimulate angiogenesis both in vivo and in vitro. However, the effect of ghrelin upon angiogenesis, and the corresponding mechanisms of ghrelin therein, in human coronary artery endothelial cells (HCAECs) under hypoxia is still unknown. Our study found that ghrelin significantly increased HCAECs proliferation, migration, in vitro angiogenesis, and microvessel sprouting from the aortic ring under hypoxic conditions. The ghrelin-induced angiogenic process was accompanied by vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and endothelial-specific receptor tyrosine kinase (Tie2) expressions. In addition, this angiogenic effect was almost completely inhibited by Ang-2 RNAi and Tie2 RNAi. Pretreatment with the GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced VEGF, Ang-1, Ang-2 and Tie2 expressions and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates HCAECs in vitro angiogenesis through GHSR1a-mediated VEGF, Ang-1, Ang-2 and Tie2 pathways under hypoxic conditions. It indicated that ghrelin might play an important role in myocardial angiogenesis after ischemic injury.
Asunto(s)
Células Endoteliales , Ghrelina , Humanos , Células Endoteliales/metabolismo , Ghrelina/farmacología , Ghrelina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vasos Coronarios , Hipoxia/metabolismo , Angiopoyetina 2/metabolismo , Angiopoyetina 2/farmacologíaRESUMEN
Purpose: The YAP signaling pathway is altered and implicated as oncogenic in human mammary cancers. However, roles of YAP signaling that regulate the breast tumor angiogenesis have remained elusive. Tumor angiogenesis is coordinated by the activation of both cancer cells and vascular endothelial cells. Whether the YAP signaling pathway can regulate the intercellular interaction between cancer cells and endothelial cells is essentially unknown. Methods: The effects of YAP on tumor angiogenesis, migration, and proliferation of vascular endothelial cells were evaluated in vitro. Expression of proteins and phosphorylating proteins involved in YAP, G13-RhoA, and PI3K/Akt signaling pathways was evaluated using the Western blotting, immunofluorescence staining, and immunohistochemistry analysis. In addition, the effects of YAP on breast cancer angiogenesis were evaluated in vivo by tumor xenograft mice. Results: We showed here that conditioned media from YAP overexpressed breast cancer cells (CM-YAP+) could promote angiogenesis, accompanied by increased tube formation, migration, and proliferation of human umbilical vein endothelial cells (HUVECs). Down regulation of YAP in HUVECs reversed CM-YAP+ induced angiogenesis. CM-YAP+ time-dependently activated YAP in HUVECs by dephosphorylating YAP and increasing nuclear translocation. We also identified that both G13-RhoA and PI3K/Akt signaling pathway were necessary for CM-YAP+ induced activation of YAP. Besides, connective tissue growth factor (CTGF) and angiopoietin-2 (ANG-2) acted as down-stream of YAP in HUVECs to promote angiogenesis. In addition, subcutaneous tumors nude mice model demonstrated that tumors overexpressed YAP revealed more neovascularization in vivo. Conclusion: YAP-YAP interaction between breast cancer cells and endothelial cells could promote tumor angiogenesis, supporting that YAP is a potential marker and target for developing novel therapeutic strategies against breast cancer.
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Angiopoyetina 2 , Neoplasias de la Mama , Proteínas de Ciclo Celular/metabolismo , Factores de Transcripción/metabolismo , Angiopoyetina 2/metabolismo , Angiopoyetina 2/farmacología , Animales , Neoplasias de la Mama/patología , Movimiento Celular , Proliferación Celular , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/farmacología , Medios de Cultivo Condicionados/farmacología , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ratones Desnudos , Neovascularización Patológica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton's jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.
Asunto(s)
Medios de Cultivo Condicionados/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Células Madre Mesenquimatosas/citología , Proteínas Recombinantes/farmacología , Cicatrización de Heridas/efectos de los fármacos , Angiopoyetina 1/metabolismo , Angiopoyetina 1/farmacología , Angiopoyetina 2/metabolismo , Angiopoyetina 2/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Medios de Cultivo Condicionados/química , Diabetes Mellitus Tipo 2/inducido químicamente , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/farmacología , Células Madre Mesenquimatosas/metabolismo , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Gelatina de Wharton/citología , Gelatina de Wharton/metabolismoRESUMEN
BACKGROUND: Sepsis in premature newborns is a risk factor for bronchopulmonary dysplasia (BPD), but underlying mechanisms of lung injury remain unclear. Aberrant expression of endothelial cell (EC) angiopoietin 2 (ANGPT2) disrupts angiopoietin 1 (ANGPT1)/TIE2-mediated endothelial quiescence, and is implicated in sepsis-induced acute respiratory distress syndrome in adults. We hypothesized that recombinant ANGPT1 will mitigate sepsis-induced ANGPT2 expression, inflammation, acute lung injury (ALI), and alveolar remodeling in the saccular lung. METHODS: Effects of recombinant ANGPT1 on lipopolysaccharide (LPS)-induced endothelial inflammation were evaluated in human pulmonary microvascular endothelial cells (HPMEC). ALI and long-term alveolar remodeling were assessed in newborn mice exposed to intraperitoneal LPS and recombinant ANGPT1 pretreatment. RESULTS: LPS dephosphorylated EC TIE2 in association with increased ANGPT2 in vivo and in vitro. ANGPT1 suppressed LPS and ANGPT2-induced EC inflammation in HPMEC. Neonatal mice treated with LPS had increased lung cytokine expression, neutrophilic influx, and cellular apoptosis. ANGPT1 pre-treatment suppressed LPS-induced lung Toll-like receptor signaling, inflammation, and ALI. LPS-induced acute increases in metalloproteinase 9 expression and elastic fiber breaks, as well as a long-term decrease in radial alveolar counts, were mitigated by ANGPT1. CONCLUSIONS: In an experimental model of sepsis-induced BPD, ANGPT1 preserved endothelial quiescence, inhibited ALI, and suppressed alveolar simplification. IMPACT: Key message: Angiopoietin 1 inhibits LPS-induced neonatal lung injury and alveolar remodeling. Additions to existing literature: Demonstrates dysregulation of angiopoietin-TIE2 axis is important for sepsis- induced acute lung injury and alveolar simplification in experimental BPD. Establishes recombinant Angiopoietin 1 as an anti-inflammatory therapy in BPD. IMPACT: Angiopoietin 1-based interventions may represent novel therapies for mitigating sepsis-induced lung injury and BPD in premature infants.
Asunto(s)
Lesión Pulmonar Aguda , Displasia Broncopulmonar , Sepsis , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/prevención & control , Angiopoyetina 1/metabolismo , Angiopoyetina 1/farmacología , Angiopoyetina 2/metabolismo , Angiopoyetina 2/farmacología , Animales , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/prevención & control , Células Endoteliales/metabolismo , Endotoxinas/metabolismo , Endotoxinas/farmacología , Humanos , Recién Nacido , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Pulmón , RatonesRESUMEN
In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and ß cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs.
Asunto(s)
Angiopoyetina 1/farmacología , Angiopoyetina 2/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Islotes Pancreáticos/metabolismo , Transducción de Señal/efectos de los fármacos , HumanosRESUMEN
Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here, we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2-induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the etiology of several complications of pregnancy.
Asunto(s)
Angiopoyetina 2/farmacología , Movimiento Celular/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Línea Celular , Femenino , Humanos , Integrinas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Fosforilación , Embarazo , Complicaciones del Embarazo/enzimología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptor TIE-2/agonistas , Receptor TIE-2/metabolismo , Trofoblastos/enzimologíaRESUMEN
Cross talk between endothelial cells and adipocytes is vital to adipocyte functions, but little is known about the mechanisms or factors controlling the process. Angiogenesis is a critical component linking the endothelium to healthy adipogenesis, yet it is not known if or how it is involved in adipocyte physiology. Therefore, the purpose of this study was to determine the effect of angiopoietin-1 (Ang-1) and -2 (Ang-2) as well as their receptor, Tie-2, on adipocyte physiology. 3T3-L1 pre- and mature adipocytes were found to express Ang-1, Ang-2, and Tie-2, which decrease upon polyunsaturated fatty acid treatment. Furthermore, 3T3-L1 cells treated with recombinant Ang-1 or Ang-2 increased expression of the antiapoptotic gene Bcl-x and decreased expression of the proapoptotic gene Casp-8. Next, preadipocytes were treated with saturated fatty acids (SFAs) to induce cell stress. SFA-mediated splicing of X-box-binding protein-1 was reduced by co-treatment with Ang-1, and cell viability was improved in the presence of SFAsâ¯+â¯Ang-1. Taken together, these results indicate that Ang-1 may protect preadipocytes from SFA-induced apoptosis and endoplasmic reticulum stress.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipogénesis , Tejido Adiposo/citología , Angiopoyetina 1/farmacología , Grasas de la Dieta/farmacología , Ácidos Grasos/farmacología , Neovascularización Fisiológica , Células 3T3-L1 , Adipocitos/metabolismo , Adipocitos/fisiología , Tejido Adiposo/irrigación sanguínea , Tejido Adiposo/fisiología , Angiopoyetina 1/metabolismo , Angiopoyetina 2/metabolismo , Angiopoyetina 2/farmacología , Animales , Apoptosis , Caspasa 8/metabolismo , Supervivencia Celular , Estrés del Retículo Endoplásmico , Células Endoteliales , Ácidos Grasos Insaturados/farmacología , Humanos , Macrófagos , Ratones , Ratones Endogámicos C57BL , Receptor TIE-2/metabolismo , Receptor TIE-2/farmacología , Proteína 1 de Unión a la X-Box/metabolismo , Proteína bcl-X/metabolismoRESUMEN
In contrast to healthy intervertebral discs (IVDs), degenerate IVDs become vascularized. Here, we determined the role of an angiogenesis promoter, angiopoietin (Ang)-2, in the pathology of IVD degeneration (IDD). We evaluated degree of IDD using the Pfirrmann grading system. We used quantitative real-time polymerase chain reaction and western blotting to analyze ANG2 gene expression and Ang-2 protein levels, respectively. The involvement of Ang-2 in IVD degradation and regulation of nuclear factor-κB (NF-κB) signaling was examined by immunohistochemistry, western blotting and immunofluorescence. As a result, 10 samples with grades II and III IDD were categorized as the mild IDD group; for comparison, another 10 specimens with grades IV and V constituted the severe IDD group. Ang-2 expression was significantly higher in severe IDD than in mild IDD. Exogenous Ang-2 administration led to increased production of catabolic proteinases and loss of aggrecan and collagen II in degenerative NP cell cultures, which was mediated by the NF-κB signaling pathway. Elevated Ang-2 levels also increased interleukin-1ß expression in degenerative NP cells. We conclude that the release of Ang-2 aggravates NP cell degradation and plays an important role in IDD. Ang-2 may thus constitute a novel therapeutic target for the treatment of IVD.
Asunto(s)
Angiopoyetina 2/farmacología , Matriz Extracelular/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Núcleo Pulposo/patología , Matriz Extracelular/efectos de los fármacos , Humanos , Inmunohistoquímica , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Cryopreservation and transplantation of ovarian tissue (OT) represents a method for fertility preservation. However, as the transplantation is performed without vessel anastomosis, unavoidable ischemic damage occurs. To reduce this ischemic damage and improve outcomes after transplantation, we used two kind of angiogenic factors, angiopoietin-2 (ang-2) and vascular endothelial growth factor (VEGF). Fresh or vitrified-warmed bovine OTs were prepared for xenotransplantation (XT). Fresh OTs were immediately xenografted into nude mice (XT-Fresh). Vitrified-warmed OTs were xenografted into four subgroups of mice, which were injected intraperitoneally before XT with saline (XT-Vitri), Ang-2 (XT-Ang-2), VEGF (XT-VEGF), and a combination of Ang-2 and VEGF (XT-Combined). Seven or 28 days post-grafting, grafted OTs and blood samples were collected for evaluation. Follicle normality was higher in the angiogenic factor-treated groups than in the XT-Vitri group. The XT-VEGF and the XT-Combined showed higher (P<0.05) follicular density than the XT-Vitri group. The highest apoptotic follicle ratio was observed in the XT-Vitri group on day 7; this was decreased (P<0.05) in the XT-Combined group. Microvessel densities were higher in the angiogenic factor-treated groups than in the XT-Vitri group. The largest fibrotic area was showed in the XT-Vitri group on day 28, and it was decreased (P<0.05) in the XT-combined group. Based on these results, administration of Ang-2 and VEGF to recipients prior to XT appeared to alleviate ischemic damage by enhancing angiogenesis, which resulted in the maintenance of follicle integrity and density, and reduced follicle apoptosis and OT fibrosis.
Asunto(s)
Angiopoyetina 2/farmacología , Apoptosis/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Folículo Ovárico , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Bovinos , Femenino , Xenoinjertos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Folículo Ovárico/irrigación sanguínea , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Folículo Ovárico/trasplanteRESUMEN
BACKGROUND: Angiopoietin-2 (Ang-2), a ligand of the Tie-2 receptor, plays an important role in maintaining endothelial cells and in destabilizing blood vessels. Collateral artery growth (arteriogenesis) is a key adaptive response to arterial occlusion. It is unknown whether the destabilization of blood vessels by Ang-2 can affect arteriogenesis and modulate mononuclear cell function. This study aimed to investigate the effects of Ang-2 on collateral artery growth. METHODS: Hindlimb ischaemia model was produced in C57BL/6 mice by femoral artery ligation. Blood flow perfusion was measured using a laser Doppler perfusion imager quantitative RT-PCR analysis was applied to identify the level of angiogenic factors. RESULTS: After the induction of hindlimb ischaemia, blood flow recovery was impaired in mice treated with recombinant Ang-2 protein; this was accompanied by a reduction of peri-collateral macrophage infiltration. In addition, quantitative RT-PCR analysis revealed that Ang-2 treatment decreased monocyte chemotactic protein-1 (MCP-1), platelet-derived growth factor-BB (PDGF-BB) mRNA levels in ischaemic adductor muscles. Ang-2 can lead to macrophage M1/M2 polarization shift inhibition in the ischaemic muscles. Furthermore, Ang-2 reduced the in vitro inflammatory response in macrophages and vascular cells involved in arteriogenesis. CONCLUSIONS: Our results demonstrate that Ang-2 is essential for efficient arteriogenesis, which controls macrophage infiltration.
Asunto(s)
Angiopoyetina 2/uso terapéutico , Arterias/crecimiento & desarrollo , Circulación Colateral , Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Isquemia/tratamiento farmacológico , Macrófagos/patología , Angiopoyetina 2/farmacología , Animales , Arterias/efectos de los fármacos , Arterias/patología , Movimiento Celular/efectos de los fármacos , Circulación Colateral/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/patología , Isquemia/genética , Isquemia/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Perfusión , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
OBJECTIVES: Retrospective studies suggest that donor desmopressin (DDAVP) treatment improves renal transplant outcome. The present study tests the hypothesis that desmopressin neutralizes the graft's endothelium from proinflammatory angiopoietin 2 containing Weibel-Palade bodies in the donor, resulting in reduced Weibel-Palade body release at the time of reperfusion in the recipient. MATERIALS AND METHODS: Using rat models, we examined the influence of desmopressin treatment on the expression of vasopressin 2 receptors and adhesion molecules in brain-dead donors, with renal function examined in allogeneic recipients. The influence of desmopressin on the expression of adhesion molecules also was tested in vitro. RESULTS: Vasopressin 2 receptors were restricted to collecting ducts and distal tubules and only scarcely found in the renal vasculature. Vasopressin 2 receptor expression was down-regulated in brain-dead rats by desmopressin. Renal expression of vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1 were significantly reduced in these rats. In contrast, angiopoietin 2 did not influence the expression of adhesion molecules in in vitro cultured endothelial cells after tumor necrosis factor ? stimulation. Donor desmopressin treatment improved neither renal function nor histology in allogeneic renal transplant recipients. CONCLUSIONS: Our data do not support the hypothesis that the clinically observed salutary effect of desmopressin is mediated by depletion of Weibel-Palade bodies in renal allografts.
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Desamino Arginina Vasopresina/farmacología , Células Endoteliales/efectos de los fármacos , Trasplante de Riñón/efectos adversos , Riñón/efectos de los fármacos , Daño por Reperfusión/prevención & control , Cuerpos de Weibel-Palade/efectos de los fármacos , Angiopoyetina 2/farmacología , Animales , Células Cultivadas , Isquemia Fría/efectos adversos , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Riñón/metabolismo , Riñón/patología , Masculino , Modelos Animales , Ratas Endogámicas F344 , Ratas Endogámicas Lew , Receptores de Vasopresinas/agonistas , Receptores de Vasopresinas/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Factores de Tiempo , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Isquemia Tibia/efectos adversos , Cuerpos de Weibel-Palade/metabolismo , Cuerpos de Weibel-Palade/patologíaRESUMEN
The homeostasis of the central nervous system is maintained by the blood-brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.
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Angiopoyetina 2/metabolismo , Barrera Hematoencefálica/fisiología , Receptor TIE-2/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal/efectos de los fármacos , Accidente Cerebrovascular/patología , Angiopoyetina 2/genética , Angiopoyetina 2/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/ultraestructura , Edema Encefálico/etiología , Edema Encefálico/patología , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/genética , Células Cultivadas , Modelos Animales de Enfermedad , Impedancia Eléctrica , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Femenino , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Transgénicos , Microvasos/citología , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Pericitos/patología , Pericitos/ultraestructura , Transducción de Señal/genética , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismoRESUMEN
The vascular leakage in diabetic retinopathy leads to macular edema and vision loss. Although astrocyte play an important role in regulating blood-brain barrier integrity in the brain, the precise role of astrocyte in blood-retinal barrier was yet to be elucidated. This study aimed to investigate the role of angiopoietin 2 (Ang2) in astrocyte loss and vascular leakage in the early streptozotocin-induced diabetic retinopathy. We demonstrated that vascular leakage occurred with astrocyte loss in early diabetic mice retina as Ang2 increased. The astrocyte loss and vascular leakage were inhibited by intravitreal injection of Ang2-neutralizing antibody. In vitro, Ang2 aggravated high glucose-induced astrocyte apoptosis via GSK-3ß activation. Ang2 directly bound to αvß5 integrin, which was abundant in astrocyte, and the blockade of αvß5 integrin, in vitro, effectively attenuated Ang2-induced astrocyte apoptosis. In vivo, intravitreal injection of anti-αvß5-integrin antibody inhibited astrocyte loss in early diabetic retinopathy. Taken together, Ang2 induced astrocyte apoptosis under high glucose via αvß5-integrin/GSK-3ß/ß-catenin pathway. Therefore, we suggest that Ang2/integrin signaling could be a potential therapeutic target to prevent the vascular leakage by astrocyte loss in early diabetic retinopathy.
Asunto(s)
Angiopoyetina 2/metabolismo , Astrocitos/metabolismo , Retinopatía Diabética/metabolismo , Receptores de Vitronectina/metabolismo , Angiopoyetina 2/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Astrocitos/patología , Retinopatía Diabética/genética , Retinopatía Diabética/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacología , Transducción de SeñalRESUMEN
Based on former studies showing an antagonism between angiopoietin-2 (Ang-2) and bacterial endotoxins (LPS), we investigated the role of Ang-2 as immunomodulatory treatment. At first, kinetics of circulating LPS in Gram-negative pyelonephritis developing after urinary obstruction was studied. Serum LPS, interleukin (IL)-6 and Ang-2 were measured in 25 patients with acute pyelonephritis and sepsis before and after removal of the obstruction performed either with insertion of a pigtail catheter (n=12) or percutaneous drainage (n=13). At a second stage, Ang-2 was given as anti-inflammatory treatment in 40 rabbits one hour after induction of acute pyelonephritis by ligation of the ureter at the level of pelvo-ureteral junction and upstream bacterial inoculation. Survival was recorded; blood mononuclear cells were isolated and stimulated for the production of tumour necrosis factor-alpha (TNFα). The decrease in circulating LPS was significantly greater among patients undergoing drainage than pigtail insertion. This was accompanied by reciprocal changes of Ang-2 and IL-6. Treatment with Ang-2 prolonged survival from Escherichia coli pyelonephritis despite high levels of circulating LPS. When Ang-2 was given as treatment of Pseudomonas aeruginosa pyelonephritis, sepsis-induced decrease of TNFα production by circulating mononuclear cells was reversed without an effect on tissue bacterial overgrowth. It is concluded that Ang-2 and LPS follow reverse kinetics in acute pyelonephritis. When given as experimental treatment, Ang-2 prolongs survival through an effect on mononuclear cells.
Asunto(s)
Angiopoyetina 2/sangre , Lipopolisacáridos/sangre , Pielonefritis/sangre , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Angiopoyetina 2/farmacología , Animales , Células Cultivadas , Escherichia coli/fisiología , Femenino , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Interleucina-6/sangre , Estimación de Kaplan-Meier , Cinética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Pseudomonas aeruginosa/fisiología , Pielonefritis/tratamiento farmacológico , Pielonefritis/microbiología , Conejos , Sepsis/sangre , Sepsis/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-ß in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-ß promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.
Asunto(s)
Angiopoyetina 1/genética , Angiopoyetina 2/genética , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide/genética , Linfoma no Hodgkin/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Enfermedad Aguda , Adolescente , Adulto , Inductores de la Angiogénesis/inmunología , Inductores de la Angiogénesis/metabolismo , Inductores de la Angiogénesis/farmacología , Angiopoyetina 1/inmunología , Angiopoyetina 1/farmacología , Angiopoyetina 2/inmunología , Angiopoyetina 2/farmacología , Antineoplásicos/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Leucemia Mieloide/inmunología , Leucemia Mieloide/patología , Leucemia Mieloide/terapia , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Linfoma no Hodgkin/terapia , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Estudios Retrospectivos , Transducción de Señal , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling.
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Anilidas/farmacología , Neovascularización Patológica/prevención & control , Neoplasias Pancreáticas/tratamiento farmacológico , Quinolinas/farmacología , Receptor TIE-2/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Angiopoyetina 2/farmacología , Animales , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Linfangiogénesis/efectos de los fármacos , Ratones Endogámicos NOD , Ratones SCID , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor C de Crecimiento Endotelial Vascular/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A key feature in the induction of pathological angiogenesis is that inflammation precedes and accompanies the formation of neovessels as evidenced by increased vascular permeability and the recruitment of inflammatory cells. Previously, we and other groups have shown that selected growth factors, namely vascular endothelial growth factor (VEGF) and angiopoietins (Ang1 and Ang2) do not only promote angiogenesis, but can also induce inflammatory response. Herein, given a pro-inflammatory environment, we addressed the individual capacity of VEGF and angiopoietins to promote the formation of mature neovessels and to identify the different types of inflammatory cells accompanying the angiogenic process over time. Sterilized polyvinyl alcohol (PVA) sponges soaked in growth factor-depleted Matrigel mixed with PBS, VEGF, Ang1, or Ang2 (200 ng/200 µl) were subcutaneously inserted into anesthetized mice. Sponges were removed at day 4, 7, 14, or 21 post-procedure for histological, immunohistological (IHC), and flow cytometry analyses. As compared to PBS-treated sponges, the three growth factors promoted the recruitment of inflammatory cells, mainly neutrophils and macrophages, and to a lesser extent, T- and B-cells. In addition, they were more potent and more rapid in the recruitment of endothelial cells (ECs) and in the formation and maturation (ensheating of smooth muscle cells around ECs) of neovessels. Thus, the autocrine/paracrine interaction among the different inflammatory cells in combination with VEGF, Ang1, or Ang2 provides a suitable microenvironment for the formation and maturation of blood vessels.
Asunto(s)
Angiopoyetinas/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Angiopoyetina 1/farmacología , Angiopoyetina 2/farmacología , Animales , Colágeno/química , Combinación de Medicamentos , Citometría de Flujo , Inmunohistoquímica , Laminina/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Fagocitosis/genética , Fagocitosis/fisiología , Proteoglicanos/químicaRESUMEN
We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.