RESUMO
OBJECTIVE: Aberrant neovascularization is a leading cause of blindness in several eye diseases, including age-related macular degeneration and proliferative diabetic retinopathy. The identification of key regulators of pathological ocular neovascularization has been a subject of extensive research and great therapeutic interest. Here, we explored the previously unrecognized role of cKIT and its ligand, SCF (stem cell factor), in the pathological ocular neovascularization process. Approach and Results: Compared with normoxia, hypoxia, a crucial driver of neovascularization, caused cKIT to be highly upregulated in endothelial cells, which significantly enhanced the angiogenic response of endothelial cells to SCF. In murine models of pathological ocular neovascularization, such as oxygen-induced retinopathy and laser-induced choroidal neovascularization models, cKIT and SCF expression was significantly increased in ocular tissues, and blockade of cKIT and SCF using cKit mutant mice and anti-SCF neutralizing IgG substantially suppressed pathological ocular neovascularization. Mechanistically, SCF/cKIT signaling induced neovascularization through phosphorylation of glycogen synthase kinase-3ß and enhancement of the nuclear translocation of ß-catenin and the transcription of ß-catenin target genes related to angiogenesis. Inhibition of ß-catenin-mediated transcription using chemical inhibitors blocked SCF-induced in vitro angiogenesis in hypoxia, and injection of a ß-catenin agonist into cKit mutant mice with oxygen-induced retinopathy significantly enhanced pathological neovascularization in the retina. Conclusions; Our data reveal that SCF and cKIT are promising novel therapeutic targets for treating vision-threatening ocular neovascular diseases.
Assuntos
Regulação da Expressão Gênica , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Neovascularização Retiniana/genética , Fator de Células-Tronco/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Inibidores da Angiogênese/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hipóxia/complicações , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/genética , Doenças Retinianas/patologia , Doenças Retinianas/fisiopatologia , Transdução de Sinais/genéticaRESUMO
Identification of differentially expressed genes in angioblasts derived from human embryonic stem cells (hESCs) is of great interest for elucidating the molecular mechanisms underlying human vasculogenesis. The aim of this study was to define hESC-derived angioblasts at the clonal level and to perform comparative transcriptional analysis to characterize their distinct gene expression profiles. In a clonal analysis performed in cell-specific differentiation media, hESC-derived CD34(+)CD31(+) cells were identified as angioblasts in that they exhibited a significantly higher ability to form endothelial cell (EC) and smooth muscle cell (SMC) colonies than CD34(+)CD31(-) and CD34(-) cell populations did. Microarray analysis showed that many genes involved in vascular development and signaling transduction were overexpressed in hESC-derived CD34(+)CD31(+) cells, whereas those related to mitosis, the DNA damage response, and translation were substantially downregulated. In addition, comparative gene expression profiling of hESC-derived CD34(+)CD31(+) cells and human somatic primary vascular cells demonstrated that hESC-derived CD34(+)CD31(+) cells expressed key genes involved in the EC and SMC differentiation processes, which supports the result that hESC-derived CD34(+)CD31(+) cells are bipotent angioblasts. Our results may provide insights into the identity and function of hESC-derived angioblasts and may also facilitate further investigation of the molecular mechanisms regulating human embryonic vasculogenesis.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica , Hemangioblastos/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Análise por Conglomerados , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Hemangioblastos/fisiologia , Humanos , Mesoderma/citologia , Mesoderma/fisiologia , Análise em Microsséries , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transcrição GênicaRESUMO
AIMS: In hypoxia, endothelial cells (ECs) proliferate, migrate, and form new vasculature in a process called angiogenesis. Recent studies have suggested that ECs rely on glycolysis to meet metabolic needs for angiogenesis in ischaemic tissues, and several studies have investigated the molecular mechanisms integrating angiogenesis and endothelial metabolism. Here, we investigated the role of stem cell factor (SCF) and its receptor, cKIT, in regulating endothelial glycolysis during hypoxia-driven angiogenesis. METHODS AND RESULTS: SCF and cKIT signalling increased the glucose uptake, lactate production, and glycolysis in human ECs under hypoxia. Mechanistically, SCF and cKIT signalling enhanced the expression of genes encoding glucose transporter 1 (GLUT1) and glycolytic enzymes via Akt- and ERK1/2-dependent increased translation of hypoxia inducible factor 1A (HIF1A). In hypoxic conditions, reduction of glycolysis and HIF-1α expression using chemical inhibitors significantly reduced the SCF-induced in vitro angiogenesis in human ECs. Compared with normal mice, mice with oxygen-induced retinopathy (OIR), characterized by ischaemia-driven pathological retinal neovascularization, displayed increased levels of SCF, cKIT, HIF-1α, GLUT1, and glycolytic enzymes in the retina. Moreover, cKIT-positive neovessels in the retina of mice with OIR showed elevated expression of GLUT1 and glycolytic enzymes. Further, blocking SCF and cKIT signalling using anti-SCF neutralizing IgG and cKIT mutant mice significantly reduced the expression of HIF-1α, GLUT1, and glycolytic enzymes and decreased the pathological neovascularization in the retina of mice with OIR. CONCLUSION: We demonstrated that SCF and cKIT signalling regulate angiogenesis by controlling endothelial glycolysis in hypoxia and elucidated the SCF/cKIT/HIF-1α axis as a novel metabolic regulation pathway during hypoxia-driven pathological angiogenesis.
Assuntos
Hipóxia Celular , Transportador de Glucose Tipo 1 , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas Proto-Oncogênicas c-kit , Transdução de Sinais , Fator de Células-Tronco , Animais , Humanos , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Endogâmicos C57BL , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Neovascularização Retiniana/genética , Camundongos , Neovascularização Fisiológica , Células Cultivadas , Modelos Animais de Doenças , Glucose/metabolismoRESUMO
BACKGROUND: Our goal was to investigate the correlation between the dysregulation of transforming growth factor-ß1 (TGF-ß1) and cystic medial degeneration in the aortic aneurysmal tissues of in Marfan syndrome (MFS) patients. Although aortic aneurysm in animal models of MFS is related to the dysregulation of TGF-ß, it has yet to be determined whether TGF-ß dysregulation correlates with pathogenic aneurysmal characteristics in MFS patients. METHODS AND RESULTS: Compared with aortic tissue from normal individuals, the medial layers of aortic tissue from MFS patients exhibited profound cystic medial degeneration and cellular apoptosis. These histopathologic changes positively correlated with the extent of TGF-ß1 signaling activation (Smad2 phosphorylation) in aneurysmal aortic tissue. In addition, the level of TGF-ß1 expression in peripheral blood and aneurysmal aortic tissues was significantly elevated in MFS patients. A significant positive correlation was observed between the plasma level of active TGF-ß1 in MFS patients and the severity of cystic medial degeneration and Smad2 phosphorylation in aneurysmal aortic medial layers. CONCLUSIONS: We found a strong association between the dysregulation of TGF-ß1 and aortic pathogenesis in human MFS patients. This suggests that the plasma concentration of TGF-ß1 in MFS patients might be a useful biomarker of the progression of aortic aneurysms.
Assuntos
Aorta/metabolismo , Aneurisma Aórtico/sangue , Síndrome de Marfan/sangue , Fator de Crescimento Transformador beta1/biossíntese , Adulto , Aorta/patologia , Aneurisma Aórtico/etiologia , Aneurisma Aórtico/patologia , Apoptose , Biomarcadores/sangue , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Síndrome de Marfan/complicações , Síndrome de Marfan/patologia , Fosforilação , Proteína Smad2/metabolismoRESUMO
To determine the role of endothelial progenitor cells (EPCs) in the pathogenesis of thromboangiitis obliterans (TAO), EPC numbers and colony-forming units, migratory function and tubular structure formation in vitro were compared between 13 young male TAO patients and two age-matched healthy control groups: 11 smokers and 12 non-smokers. TAO patients had significantly lower numbers of EPCs and EPC colonies compared to both non-smokers [190 (97.0-229) vs 528 (380-556), p < 0.001 for EPCs and 0.80 (0.53-1.00) vs 2.80 (2.08-4.00) per mm(2), p = 0.001 for EPC colonies] and smokers [190 (97.0-229) vs 272 (229-326), p = 0.012 for EPCs and 0.80 (0.53-1.00) vs 2.80 (1.80-3.93) per mm(2), p = 0.001 for EPC colonies]. However, there were no significant differences in migratory function or tube formation between the three groups. These results suggest that TAO patients have an intrinsic decrease in EPCs not entirely associated with smoking, which may be the cause of endothelial dysfunction seen in TAO patients leading to the development of this disease at early ages.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/citologia , Células-Tronco/citologia , Tromboangiite Obliterante/patologia , Adulto , Diferenciação Celular/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fumar/efeitos adversos , Tromboangiite Obliterante/metabolismo , Adulto JovemRESUMO
Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.
Assuntos
Angiopoietina-2/metabolismo , Endotélio Vascular/metabolismo , Vasos Linfáticos/metabolismo , Receptor de TIE-1/metabolismo , Receptor TIE-2/agonistas , Animais , Células Cultivadas , Endotélio Vascular/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Vasos Linfáticos/enzimologia , Camundongos , Receptor TIE-2/metabolismoRESUMO
RATIONALE: Generation of induced pluripotent stem (iPS) cells has been intensively studied by a variety of reprogramming methods, but the molecular and functional properties of the cells differentiated from iPS cells have not been well characterized. OBJECTIVE: To address this issue, we generated iPS cells from human aortic vascular smooth muscle cells (HASMCs) using lentiviral transduction of defined transcription factors and differentiated these iPS cells back into smooth muscle cells (SMCs). METHODS AND RESULTS: Established iPS cells were shown to possess properties equivalent to human embryonic stem cells, in terms of the cell surface markers, global mRNA and microRNA expression patterns, epigenetic status of OCT4, REX1, and NANOG promoters, and in vitro/in vivo pluripotency. The cells were differentiated into SMCs to enable a direct, comparative analysis with HASMCs, from which the iPS cells originated. We observed that iPS cell-derived SMCs were very similar to parental HASMCs in gene expression patterns, epigenetic modifications of pluripotency-related genes, and in vitro functional properties. However, the iPS cells still expressed a significant amount of lentiviral transgenes (OCT4 and LIN28) because of partial gene silencing. CONCLUSIONS: Our study reports, for the first time, the generation of iPS cells from HASMCs and their differentiation into SMCs. Moreover, a parallel comparative analysis of human iPS cell-derived SMCs and parental HASMCs revealed that iPS-derived cells possessed representative molecular and in vitro functional characteristics of parental HASMCs, suggesting that iPS cells hold great promise as an autologous cell source for patient-specific cell therapy.
Assuntos
Aorta/metabolismo , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/citologia , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Lentivirus , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Miócitos de Músculo Liso/citologia , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Transdução GenéticaRESUMO
Pulmonary arterial hypertension (PAH) is a progressive and devastating disease whose pathogenesis is associated with a phenotypic switch of pulmonary arterial vascular smooth muscle cells (PASMCs). Bone morphogenetic protein (BMP) signaling and potassium two pore domain channel subfamily K member 3 (KCNK3) play crucial roles in PAH pathogenesis. However, the relationship between BMP signaling and KCNK3 expression in the PASMC phenotypic switching process has not been studied. In this study, we explored the effect of BMPs on KCNK3 expression and the role of KCNK3 in the BMP-mediated PASMC phenotypic switch. Expression levels of BMP receptor 2 (BMPR2) and KCNK3 were downregulated in PASMCs of rats with PAH compared to those in normal controls, implying a possible association between BMP/BMPR2 signaling and KCNK3 expression in the pulmonary vasculature. Treatment with BMP2, BMP4, and BMP7 significantly increased KCNK3 expression in primary human PASMCs (HPASMCs). BMPR2 knockdown and treatment with Smad1/5 signaling inhibitor substantially abrogated the BMP-induced increase in KCNK3 expression, suggesting that KCNK3 expression in HPASMCs is regulated by the canonical BMP-BMPR2-Smad1/5 signaling pathway. Furthermore, KCNK3 knockdown and treatment with a KCNK3 channel blocker completely blocked BMP-mediated anti-proliferation and expression of contractile marker genes in HPAMSCs, suggesting that the expression and functional activity of KCNK3 are required for BMP-mediated acquisition of the quiescent PASMC phenotype. Overall, our findings show a crosstalk between BMP signaling and KCNK3 in regulating the PASMC phenotype, wherein BMPs upregulate KCNK3 expression and KCNK3 then mediates BMP-induced phenotypic switching of PASMCs. Our results indicate that the dysfunction and/or downregulation of BMPR2 and KCNK3 observed in PAH work together to induce aberrant changes in the PASMC phenotype, providing insights into the complex molecular pathogenesis of PAH. [BMB Reports 2022; 55(11): 565-570].
Assuntos
Hipertensão Pulmonar , Músculo Liso Vascular , Proteínas do Tecido Nervoso , Canais de Potássio de Domínios Poros em Tandem , Animais , Humanos , Ratos , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células , Células Cultivadas , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Transdução de Sinais , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas do Tecido Nervoso/genéticaRESUMO
Diabetic retinopathy is the leading cause of blindness which is associated with excessive angiogenesis. Using the structure of wondonin marine natural products, we previously created a scaffold to develop a novel type of antiangiogenesis agent that possesses minimized cytotoxicity. To overcome its poor pharmaceutical properties, we further modified the structure. A new scaffold was derived in which the stereogenic carbon was changed to nitrogen and the 1,2,3-triazole ring was replaced by an alkyl chain. By comparing the bioactivity versus cytotoxicity, compound 31 was selected, which has improved aqueous solubility and an enhanced selectivity index. Mechanistically, 31 suppressed angiopoietin-2 (ANGPT2) expression induced by high glucose in retinal cells and exhibited in vivo antiangiogenic activity in choroidal neovascularization and oxygen-induced retinopathy mouse models. These results suggest the potential of 31 as a lead to develop antiangiogenic small-molecule drugs to treat diabetic retinopathy and as a chemical tool to elucidate new mechanisms of angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Desenho de Fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/uso terapêutico , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Estabilidade de Medicamentos , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.
RESUMO
Dipeptidyl peptidase-4 (DPP-4) inhibitors are used for the treatment of type 2 diabetes mellitus (DM). Recent studies have shown that beyond their effect in lowing glucose, DPP-4 inhibitors mitigate DM-related microvascular complications, such as diabetic retinopathy. However, the mechanism by which pathological retinal neovascularization, a major clinical manifestation of diabetic retinopathy, is inhibited is unclear. This study sought to examine the effects of evogliptin, a potent DPP-4 inhibitor, on pathological retinal neovascularization in mice and elucidate the mechanism by which evogliptin inhibits angiogenesis mediated by vascular endothelial growth factor (VEGF), a key factor in the vascular pathogenesis of proliferative diabetic retinopathy (PDR). In a murine model of PDR, an intravitreal injection of evogliptin significantly suppressed aberrant retinal neovascularization. In human endothelial cells, evogliptin reduced VEGF-induced angiogenesis. Western blot analysis showed that evogliptin inhibited the phosphorylation of signaling molecules associated with VEGF-induced cell adhesion and migration. Moreover, evogliptin substantially inhibited the VEGF-induced activation of adenosine 5'-diphosphate ribosylation factor 6 (Arf6), a small guanosine 5'-triphosphatase (GTPase) that regulates VEGF receptor 2 signal transduction. Direct activation of Arf6 using a chemical inhibitor of Arf-directed GTPase-activating protein completely abrogated the inhibitory effect of evogliptin on VEGF-induced activation of the angiogenic signaling pathway, which suggests that evogliptin suppresses VEGF-induced angiogenesis by blocking Arf6 activation. Our results provide insights into the molecular mechanism of the direct inhibitory effect of the DPP-4 inhibitor evogliptin on pathological retinal neovascularization. In addition to its glucose-lowering effect, the antiangiogenic effect of evogliptin could also render it beneficial for individuals with PDR.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Piperazinas/farmacologia , Neovascularização Retiniana/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Inibidores da Angiogênese/farmacologia , Animais , Modelos Animais de Doenças , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Fosforilação , Neovascularização Retiniana/tratamento farmacológico , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 (Fbn1) gene, has vascular manifestations including aortic aneurysm, dissection, and rupture. Its vascular pathogenesis is assumed to be attributed to increased transforming growth factor ß (TGFß) signaling and blockade of excessive TGFß signaling has been thought to prevent dissection and aneurysm formation. Here, we investigated whether galunisertib, a potent small-molecule inhibitor of TGFß receptor I (TßRI), attenuates aneurysmal disease in a murine model of MFS (Fbn1C1039G/+) and compared the impact of galuninsertib on the MFS-related vascular pathogenesis with that of losartan, a prophylactic agent routinely used for patients with MFS. Fbn1C1039G/+ mice were administered galunisertib or losartan for 8 weeks, and their ascending aortas were assessed for histopathological changes and phosphorylation of Smad2 and extracellular signal-regulated kinase 1/2 (Erk1/2). Mice treated with galunisertib or losartan barely exhibited phosphorylated Smad2, suggesting that both drugs effectively blocked overactivated canonical TGFß signaling in Fbn1C1039G/+ mice. However, galunisertib treatment did not attenuate disrupted medial wall architecture and only partially decreased Erk1/2 phosphorylation, whereas losartan significantly inhibited MFS-associated aortopathy and markedly decreased Erk1/2 phosphorylation in Fbn1C1039G/+ mice. These data unexpectedly revealed that galunisertib, a TßRI inhibitor, showed no benefits in aneurysmal disease in MFS mice although it completely blocked Smad2 phosphorylation. The significant losartaninduced inhibition of both aortic vascular pathogenesis and Smad2 phosphorylation implied that canonical TGFß signaling might not prominently drive aneurysmal diseases in MFS mice.
RESUMO
Loss of BMP (bone morphogenic protein) signaling induces a phenotype switch of pulmonary arterial smooth muscle cells (PASMCs), which is the pathological basis of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Here, we identified FGF12 (fibroblast growth factor 12) as a novel regulator of the BMP-induced phenotype change in PASMCs and elucidated its role in pulmonary vascular remodeling during PAH development. Using murine models of PAH and lung specimens of patients with PAH, we observed that FGF12 expression was significantly reduced in PASMCs. In human PASMCs, FGF12 expression was increased by canonical BMP signaling. FGF12 knockdown blocked the antiproliferative and prodifferentiation effect of BMP on human PASMCs, suggesting that FGF12 is required for the BMP-mediated acquisition of the quiescent and differentiated PASMC phenotype. Mechanistically, FGF12 regulated the BMP-induced phenotype change by inducing MEF2a (myocyte enhancer factor 2a) phosphorylation via p38MAPK signaling, thereby modulating the expression of MEF2a target genes involved in cell proliferation and differentiation. Furthermore, we observed that TG (transgenic) mice with smooth muscle cell-specific FGF12 overexpression were protected from chronic hypoxia-induced PAH development, pulmonary vascular remodeling, and right ventricular hypertrophy. Consistent with the in vitro data using human PASMCs, FGF12 TG mice showed increased MEF2a phosphorylation and a substantial change in MEF2a target gene expression, compared with the WT (wild type) controls. Overall, our findings demonstrate a novel BMP/FGF12/MEF2a pathway regulating the PASMC phenotype switch and suggest FGF12 as a potential target for the development of therapeutics for ameliorating pulmonary vascular remodeling in PAH.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Remodelação Vascular/genética , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células/genética , Células Cultivadas , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To evaluate the efficacy of the curcumin-coating stent (CCS) on the inhibition of restenosis in a rabbit iliac artery stent model. RESULTS: Curcumin, pigment naturally acquired from the rhizome of the plant curcuma longa, is known to have antiproliferative, antimigratory, and anti-inflammatory effects. However, it is still unclear that curcumin can inhibit neointimal proliferation of the injured vessel. METHODS: Dose-dependent inhibition of cell growth was observed over a dose range from 10 nM to 10 microM. CCS was prepared by a dip-coating method (high-dose: HD, low-dose: LD). The release profile of the HD CCS showed that drug release persisted until day 21. Scanning electron microscopy of the CCS showed an intact surface of the stent even after expansion. To test the efficacy of CCS in vivo, LD CCS, HD CCS, and bare metal stents (BMS) were implanted in random order in one iliac artery (N = 30 arteries) of male New Zealand White rabbits (N = 15). RESULTS: After 28 days, the LD and HD CCS groups had a 43% and 55% reduction in the neointimal area, compared with the BMS group (BMS 3.3 +/- 1.0 mm(2), LD 1.9 +/- 0.8 mm(2), and HD 0.9 +/- 0.5 mm(2), P < 0.05). There appeared to be no cytotoxicity related to curcumin at the indicated doses. CONCLUSIONS: Curcumin, a natural compound in the human diet, seems to be a safe and effective candidate drug for use in a drug-eluting stent for the prevention of stent restenosis following angioplasty.
Assuntos
Angioplastia com Balão/instrumentação , Arteriopatias Oclusivas/prevenção & controle , Fármacos Cardiovasculares/administração & dosagem , Materiais Revestidos Biocompatíveis , Curcumina/administração & dosagem , Stents Farmacológicos/efeitos adversos , Hipercolesterolemia/complicações , Artéria Ilíaca/efeitos dos fármacos , Angioplastia com Balão/efeitos adversos , Animais , Arteriopatias Oclusivas/etiologia , Arteriopatias Oclusivas/patologia , Becaplermina , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Constrição Patológica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Artéria Ilíaca/patologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Desenho de Prótese , Proteínas Proto-Oncogênicas c-sis , Coelhos , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Fatores de TempoRESUMO
AIMS: The endothelium has emerged recently as a therapeutic target in the treatment of hypertension because endothelial dysfunction and subsequent vascular rarefaction cause target organ damage and further elevate blood pressure (BP). It led us to hypothesize that one of the endothelial survival factors, a potent derivative of angiopoietin-1 (cartilage oligomeric matrix protein, COMP-Ang-1), could be a novel class of antihypertensive agents that maintain endothelial integrity and function, thereby preventing the development of hypertension and target organ damage. METHODS AND RESULTS: To study the role of COMP-Ang-1 in preventing hypertension and target organ damage, a COMP-Ang-1 plasmid was electroporated into adductor muscles of 6 weeks old, pre-hypertensive, spontaneously hypertensive rats (SHRs), and the secretion of its expressed protein into the bloodstream was confirmed by western blotting. In comparison with sham and reporter gene transfer, COMP-Ang-1 gene transfer significantly prevented increases in systolic BP and reduced microvascular rarefaction and tissue damage in the heart and kidney. However, overexpression of soluble Tie2 receptor completely abolished these beneficial effects of COMP-Ang-1 gene transfer on SHRs, indicating that expressed COMP-Ang-1 protein has antihypertensive effects in SHRs by binding Tie2 receptors on the vascular endothelium. In particular, COMP-Ang-1 gene-transferred SHRs had significantly higher plasma levels of nitrite than other controls, which was found to be due to that expressed COMP-Ang-1 protein promoted nitrite synthesis by activating endothelial nitric oxide synthase, one of the Tie2 downstream-signalling molecules. CONCLUSION: The present study suggests a new potential of endothelial survival factor, COMP-Ang-1, as an antihypertensive agent that effectively reduces the hypertension-associated cardiovascular and renal damage, as well as prevents the further elevation of BP.
Assuntos
Angiopoietina-1/metabolismo , Anti-Hipertensivos/metabolismo , Eletroquimioterapia , Endotélio Vascular/metabolismo , Terapia Genética/métodos , Hipertensão/prevenção & controle , Receptor TIE-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Angiopoietina-1/genética , Animais , Pressão Sanguínea , Capilares/metabolismo , Capilares/patologia , Modelos Animais de Doenças , Progressão da Doença , Endotélio Vascular/enzimologia , Endotélio Vascular/patologia , Ativação Enzimática , Técnicas de Transferência de Genes , Cardiopatias/etiologia , Cardiopatias/metabolismo , Cardiopatias/prevenção & controle , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/prevenção & controle , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III , Nitritos/sangue , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Proteínas Recombinantes de Fusão/genéticaRESUMO
OBJECTIVE: Ischemia-dependent upregulation of angiopoietin2 (Ang2) led us to hypothesize the potentially proangiogenic Ang2-Tie2 signaling in endothelial progenitor cells (EPCs). Given the well-known vascular destabilizing action of Ang2 in mature endothelium, we investigated the yet unidentified mechanism behind cell-dependent differential activity of Ang2. METHODS AND RESULTS: Both in vitro and in vivo experiments showed that Ang2 promoted angiogenicity of human cord blood-derived EPCs, where Ang2 directly activated Tie2 and its related downstream signaling molecules. However, Ang2 had no such effect in fully differentiated human umbilical vein endothelial cells (HUVECs) under the same condition. Such a cell-dependent Tie2 activation by Ang2 was explained by comparing EPCs and HUVECs, where most Tie2 receptors in EPCs were found to be present unbound to Tie1, whereas those in HUVECs existed as heterocomplexes with Tie1. When Tie2 in HUVECs was prevented from forming heterocomplexes by silencing Tie1 expression, they underwent rapid phosphorylation upon Ang2 treatment, as shown in EPCs. CONCLUSIONS: In contrast with its roles in mature endothelial cells, Ang2 has proangiogenic activities in EPC directly through Tie2 signaling pathway. Such a cell-dependent differential reactivity of Ang2 was for the first time found to be modulated by physical association between Tie1 and Tie2, which inhibited Ang2-mediated Tie2 activation.
Assuntos
Angiopoietina-2/metabolismo , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptores de TIE/metabolismo , Células-Tronco/metabolismo , Regulação para Cima , Western Blotting/métodos , Células Cultivadas , Humanos , Imuno-Histoquímica , Imunoprecipitação , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptor de TIE-1/metabolismo , Receptor TIE-2/metabolismo , Receptores de TIE/genética , Veias UmbilicaisRESUMO
Purpose: Vascular endothelial growth factor (VEGF) signaling via VEGF receptor 2 (VEGFR2) plays a crucial role in pathologic ocular neovascularization. In this study, we investigated the antiangiogenic effect of apatinib, a pharmacologic inhibitor of VEGFR2 tyrosine kinase, against oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) in mice. Methods: Western blotting and in vitro angiogenesis assays were performed using human retinal microvascular endothelial cells (HRMECs). OIR was induced in neonatal mice by exposure to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17. Experimental CNV was induced in mice using laser photocoagulation. Apatinib was intravitreally and orally administered to mice. Neovascularization and phosphorylation of VEGFR2 were evaluated by immunofluorescence staining. Results: Apatinib inhibited VEGF-mediated activation of VEGFR2 signaling and substantially reduced VEGF-induced proliferation, migration, and cord formation in HRMECs. A single intravitreal injection of apatinib significantly attenuated retinal or choroidal neovascularization in mice with OIR or laser injury-induced CNV, respectively. Retinal or choroidal tissues of the eyes treated with apatinib exhibited substantially lower phosphorylation of VEGFR2 than those of controls injected with vehicle. Intravitreal injection of apatinib did not cause noticeable ocular toxicity. Moreover, oral administration of apatinib significantly reduced laser-induced CNV in mice. Conclusions: Our study demonstrates that apatinib inhibits pathologic ocular neovascularization in mice with OIR or laser-induced CNV. Apatinib may, therefore, be a promising drug for the prevention and treatment of ischemia-induced proliferative retinopathy and neovascular age-related macular degeneration.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Modelos Animais de Doenças , Piridinas/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Oral , Animais , Animais Recém-Nascidos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Células Endoteliais/efeitos dos fármacos , Feminino , Angiofluoresceinografia , Técnica Indireta de Fluorescência para Anticorpo , Injeções Intravítreas , Fotocoagulação a Laser , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Fosforilação , Gravidez , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pathological angiogenesis is one of the major symptoms of severe ocular diseases, including corneal neovascularization. The blockade of vascular endothelial growth factor (VEGF) action has been recognized as an efficient strategy for treating corneal neovascularization. In this study, we aimed to investigate whether nanoparticle-based delivery of apatinib, a novel and selective inhibitor of VEGF receptor 2, inhibits VEGF-mediated angiogenesis and suppresses experimental corneal neovascularization. Water-insoluble apatinib was encapsulated in nanoparticles composed of human serum albumin (HSA)-conjugated polyethylene glycol (PEG). In vitro angiogenesis assays showed that apatinib-loaded HSA-PEG (Apa-HSA-PEG) nanoparticles potently inhibited VEGF-induced tube formation, scratch wounding migration, and proliferation of human endothelial cells. In a rat model of alkali burn injury-induced corneal neovascularization, a subconjunctival injection of Apa-HSA-PEG nanoparticles induced a significant decrease in neovascularization compared to that observed with an injection of free apatinib solution or phosphate-buffered saline. An in vivo distribution study using HSA-PEG nanoparticles loaded with fluorescent hydrophobic model drugs revealed the presence of a substantial number of nanoparticles in the corneal stroma within 24 h after injection. These in vitro and in vivo results demonstrate that apatinib-loaded nanoparticles may be promising for the prevention and treatment of corneal neovascularization-related ocular disorders.
Assuntos
Indutores da Angiogênese/administração & dosagem , Neovascularização da Córnea/tratamento farmacológico , Nanopartículas/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Piridinas/administração & dosagem , Indutores da Angiogênese/farmacologia , Animais , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Nanopartículas/química , Polietilenoglicóis/química , Piridinas/farmacologia , Ratos Sprague-Dawley , Albumina Sérica/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Pulmonary hypertension (PH) is characterized by structural and functional changes in the lung including proliferation of vascular smooth muscle cells (VSMCs) and excessive collagen synthesis. Although connective tissue growth factor (CTGF) is known to promote cell proliferation, migration, adhesion, and extracellular matrix production in various tissues, studies on the role of CTGF in pulmonary hypertension have been limited. Here, we examined CTGF expression in the lung tissues of male Sprague Dawley rats treated with monocrotaline (MCT, 60 microg/kg), a pneumotoxic agent known to induce PH in animals. Establishment of PH was verified by the significantly increased right ventricular systolic pressure and right ventricle/left ventricle weight ratio in the MCT-treated rats. Histological examination of the lung revealed profound muscular hypertrophy in the media of pulmonary artery and arterioles in MCT-treated group. Lung parenchyma, vein, and bronchiole did not appear to be affected. RT-PCR analysis of the lung tissue at 5 weeks indicated significantly increased expression of CTGF in the MCT-treated group. In situ hybridization studies also confirmed abundant CTGF mRNA expression in VSMCs of the arteries and arterioles, clustered pneumocytes, and infiltrated macrophages. Interestingly, CTGF mRNA was not detected in VSMCs of vein or bronchiole. In saline-injected control, basal expression of CTGF was seen in bronchial epithelial cells, alveolar lining cells, and endothelial cells. Taken together, our results suggest that CTGF upregulation in arterial VSMC of the lung might be important in the pathogenesis of pulmonary hypertension. Antagonizing the role of CTGF could thus be one of the potential approaches for the treatment of PH.
Assuntos
Hipertensão Pulmonar/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/metabolismo , Monocrotalina/toxicidade , Animais , Pressão Sanguínea/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Pulmão/citologia , Pulmão/efeitos dos fármacos , Masculino , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
OBJECTIVE: Increased risk of cardiovascular disease in patients with chronic renal failure (CRF) has been explained by accelerated atherosclerosis and impaired angiogenesis, in which endothelial progenitor cells (EPCs) may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF. METHODS AND RESULTS: EPCs were isolated from CRF patients on maintenance hemodialysis (n=44) and from a normal control group (n=30). CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared with the controls (P<0.001). These findings were corroborated by 30.5% decrease in EPC migratory function in response to vascular endothelial growth factor (VEGF) (P=0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) (P<0.001). In addition, Framingham's risk factor score of both CRF (r=-0.461, P=0.010) and normal group (r=-0.367, P=0.016) significantly correlated with the numbers of EPC. Indeed, the number of circulating EPC was significantly lower in CRF patients than in normal group under the same burden of risk factors (P<0.001). A significant correlation was also observed between dialysis dose (Kt/V) and EPC incorporation into HUVEC (r=0.427, P=0.004). CONCLUSIONS: EPC biology, which is critical for neovascularization and the maintenance of vascular function, is altered in CRF. Our data strongly suggest that dysfunction of circulating EPC has a role in the progression of cardiovascular disease in patients with CRF.