RESUMO
Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1alpha gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.
Assuntos
Envelhecimento/metabolismo , Movimento Celular/fisiologia , Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/genética , Isquemia/terapia , Extremidade Inferior/irrigação sanguínea , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Envelhecimento/genética , Envelhecimento/patologia , Animais , Movimento Celular/genética , Células Cultivadas , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/uso terapêutico , Isquemia/metabolismo , Isquemia/patologia , Extremidade Inferior/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neovascularização Patológica/metabolismo , Reperfusão/métodosRESUMO
Autophagy is a process by which cytoplasmic organelles can be catabolized either to remove defective structures or as a means of providing macromolecules for energy generation under conditions of nutrient starvation. In this study we demonstrate that mitochondrial autophagy is induced by hypoxia, that this process requires the hypoxia-dependent factor-1-dependent expression of BNIP3 and the constitutive expression of Beclin-1 and Atg5, and that in cells subjected to prolonged hypoxia, mitochondrial autophagy is an adaptive metabolic response which is necessary to prevent increased levels of reactive oxygen species and cell death.
Assuntos
Autofagia , Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Mitocôndrias/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Morte Celular , Citoplasma/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Mitocondriais/biossíntese , Modelos Biológicos , Conformação Molecular , Proteínas/metabolismo , Espécies Reativas de OxigênioRESUMO
Hypoxia-inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis that controls the expression of genes encoding proteins that play key roles in angiogenesis, erythropoiesis, and glucose/energy metabolism. The stability of the HIF-1alpha subunit is regulated by ubiquitination and proteasomal degradation. In aerobic cells, O(2)-dependent prolyl hydroxylation of HIF-1alpha is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, which then recruits the Elongin C ubiquitin-ligase complex. SSAT2 (spermidine/spermine N-acetyltransferase-2) binds to HIF-1alpha and promotes its ubiquitination/degradation by stabilizing the interaction of VHL and Elongin C. Treatment of cells with heat shock protein HSP90 inhibitors induces the degradation of HIF-1alpha even under hypoxic conditions. HSP90 competes with RACK1 for binding to HIF-1alpha, and HSP90 inhibition leads to increased binding of RACK1, which recruits the Elongin C ubiquitin-ligase complex to HIF-1alpha in an O(2)-independent manner. In this work, we demonstrate that SSAT1, which shares 46% amino acid identity with SSAT2, also binds to HIF-1alpha and promotes its ubiquitination/degradation. However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1alpha with RACK1. Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O(2)-independent and O(2)-dependent degradation of HIF-1alpha.
Assuntos
Acetiltransferases/fisiologia , Proteínas de Ligação ao GTP/química , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Neoplasias/química , Receptores de Superfície Celular/química , Ubiquitina/química , Acetiltransferases/metabolismo , Elonguina , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Hipóxia , Modelos Biológicos , Oxigênio/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/metabolismo , Receptores de Quinase C Ativada , Fatores de Transcrição/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that functions as a master regulator of oxygen homeostasis. The HIF-1alpha subunit is subjected to O(2)-dependent prolyl hydroxylation leading to ubiquitination by the von Hippel-Lindau protein (VHL)-Elongin C ubiquitin-ligase complex and degradation by the 26 S proteasome. In this study, we demonstrate that spermidine/spermine-N(1)-acetyltransferase (SSAT) 2 plays an essential role in this process. SSAT2 binds to HIF-1alpha, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1alpha by stabilizing the interaction of VHL and Elongin C. Multivalent interactions by SSAT2 provide a mechanism to ensure efficient complex formation, which is necessary for the extremely rapid ubiquitination and degradation of HIF-1alpha that is observed in oxygenated cells.