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1.
Development ; 151(11)2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38770916

RESUMEN

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.


Asunto(s)
Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Endoteliales , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Receptores Notch , Neovascularización Retiniana , Transducción de Señal , Regulación hacia Arriba , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones , Receptores Notch/metabolismo , Receptores Notch/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Células Endoteliales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Vasos Retinianos/metabolismo , Angiogénesis
2.
Development ; 148(23)2021 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-34874450

RESUMEN

Under normoxia, hypoxia inducible factor (HIF) α subunits are hydroxylated by PHDs (prolyl hydroxylase domain proteins) and subsequently undergo polyubiquitylation and degradation. Normal embryogenesis occurs under hypoxia, which suppresses PHD activities and allows HIFα to stabilize and regulate development. In this Primer, we explain molecular mechanisms of the oxygen-sensing pathway, summarize HIF-regulated downstream events, discuss loss-of-function phenotypes primarily in mouse development, and highlight clinical relevance to angiogenesis and tissue repair.


Asunto(s)
Embrión de Mamíferos/embriología , Desarrollo Embrionario , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/metabolismo , Ubiquitinación , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones
3.
Development ; 146(8)2019 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-30910827

RESUMEN

Vascular pruning is crucial for normal development, but its underlying mechanisms are poorly understood. Here, we report that retinal vascular pruning is controlled by the oxygen-sensing mechanism in local astrocytes. Oxygen sensing is mediated by prolyl hydroxylase domain proteins (PHDs), which use O2 as a substrate to hydroxylate specific prolyl residues on hypoxia inducible factor (HIF)-α proteins, labeling them for polyubiquitylation and proteasomal degradation. In neonatal mice, astrocytic PHD2 deficiency led to elevated HIF-2α protein levels, expanded retinal astrocyte population and defective vascular pruning. Although astrocytic VEGF-A was also increased, anti-VEGF failed to rescue vascular pruning. However, stimulation of retinal astrocytic growth by intravitreal delivery of PDGF-A was sufficient to block retinal vascular pruning in wild-type mice. We propose that in normal development, oxygen from nascent retinal vasculature triggers PHD2-dependent HIF-2α degradation in nearby astrocytic precursors, thus limiting their further growth by driving them to differentiate into non-proliferative mature astrocytes. The physiological limit of retinal capillary density may be set by astrocytes available to support their survival, with excess capillaries destined for regression.This article has an associated 'The people behind the papers' interview.


Asunto(s)
Astrocitos/citología , Astrocitos/metabolismo , Oxígeno/metabolismo , Retina/citología , Retina/metabolismo , Animales , Apoptosis/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ratones , Seudópodos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
4.
PLoS Genet ; 15(12): e1008468, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31877123

RESUMEN

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease in which the dystrophin coding for a membrane stabilizing protein is mutated. Recently, the vasculature has also shown to be perturbed in DMD and DMD model mdx mice. Recent DMD transcriptomics revealed the defects were correlated to a vascular endothelial growth factor (VEGF) signaling pathway. To reveal the relationship between DMD and VEGF signaling, mdx mice were crossed with constitutive (CAGCreERTM:Flt1LoxP/LoxP) and endothelial cell-specific conditional gene knockout mice (Cdh5CreERT2:Flt1LoxP/LoxP) for Flt1 (VEGFR1) which is a decoy receptor for VEGF. Here, we showed that while constitutive deletion of Flt1 is detrimental to the skeletal muscle function, endothelial cell-specific Flt1 deletion resulted in increased vascular density, increased satellite cell number and improvement in the DMD-associated phenotype in the mdx mice. These decreases in pathology, including improved muscle histology and function, were recapitulated in mdx mice given anti-FLT1 peptides or monoclonal antibodies, which blocked VEGF-FLT1 binding. The histological and functional improvement of dystrophic muscle by FLT1 blockade provides a novel pharmacological strategy for the potential treatment of DMD.


Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Distrofia Muscular de Duchenne/tratamiento farmacológico , Péptidos/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Especificidad de Órganos , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética
5.
Dev Biol ; 459(2): 65-71, 2020 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-31790655

RESUMEN

Vascular endothelial growth factor (VEGF) is a potent mitogen critical for angiogenesis and organogenesis. Deletion or inhibition of VEGF during development not only profoundly suppresses vascular outgrowth, but significantly affects the development and function of various organs. In the brain, VEGF is thought to not only promote vascular growth, but also directly act on neurons as a neurotrophic factor by activating VEGF receptors. In the present study, we demonstrated that deletion of VEGF using hGfap-Cre line, which recombines genes specifically in cortical and hippocampal neurons, severely impaired brain organization and vascularization of these regions. The mutant mice had motor deficits, with lethality around the time of weaning. Multiple reporter lines indicated that VEGF was highly expressed in neurons, but that its cognate receptors, VEGFR1 and 2 were exclusive to endothelial cells in the brain. In accordance, mice lacking neuronal VEGFR1 and VEGFR2 did not exhibit neuronal deformities or lethality. Taken together, our data suggest that neuron-derived VEGF contributes to cortical and hippocampal development likely through angiogenesis independently of direct neurotrophic effects mediated by VEGFR1 and 2.


Asunto(s)
Hipocampo/crecimiento & desarrollo , Neuronas/metabolismo , Lóbulo Parietal/crecimiento & desarrollo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Alelos , Animales , Células Endoteliales/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Neovascularización Fisiológica/genética , Reacción en Cadena de la Polimerasa , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
6.
J Am Soc Nephrol ; 27(2): 428-38, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26054543

RESUMEN

Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.


Asunto(s)
Hipoxia de la Célula/fisiología , Eritropoyetina/biosíntesis , Riñón/citología , Miofibroblastos/metabolismo , Animales , Ratones , Transducción de Señal
7.
J Biol Chem ; 290(33): 20580-9, 2015 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-26124271

RESUMEN

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.


Asunto(s)
Mucosa Intestinal/fisiología , Ocludina/fisiología , Procolágeno-Prolina Dioxigenasa/fisiología , Animales , Colitis/genética , Colitis/prevención & control , Eliminación de Gen , Células HEK293 , Humanos , Ratones , Ratones Transgénicos
8.
Am J Pathol ; 184(4): 1240-1250, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24508125

RESUMEN

Prolyl hydroxylase domain (PHD) proteins catalyze oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor 1α and 2α, tagging them for pVHL-dependent polyubiquitination and proteasomal degradation. In this study, albumin Cre (Alb(Cre))-mediated, hepatocyte-specific triple disruption of Phd1, Phd2, and Phd3 (Phd(1/2/3)hKO) promoted liver erythropoietin (EPO) expression 1246-fold, whereas renal EPO was down-regulated to 6.7% of normal levels. In Phd(1/2/3)hKO mice, hematocrit levels reached 82.4%, accompanied by severe vascular malformation and steatosis in the liver. In mice double-deficient for hepatic PHD2 and PHD3 (Phd(2/3)hKO), liver EPO increase and renal EPO loss both occurred but were much less dramatic than in Phd(1/2/3)hKO mice. Hematocrit levels, vascular organization, and liver lipid contents all appeared normal in Phd(2/3)hKO mice. In a chronic renal failure model, Phd(2/3)hKO mice maintained normal hematocrit levels throughout the 8-week time course, whereas floxed controls developed severe anemia. Maintenance of normal hematocrit levels in Phd(2/3)hKO mice was accomplished by sensitized induction of liver EPO expression. Consistent with such a mechanism, liver HIF-2α accumulated to higher levels in Phd(2/3)hKO mice in response to conditions causing modest systemic hypoxia. Besides promoting erythropoiesis, EPO is also known to modulate retinal vascular integrity and neovascularization. In Phd(1/2/3)hKO mice, however, neonatal retinas remained sensitive to oxygen-induced retinopathy, suggesting that local EPO may be more important than hepatic and/or renal EPO in mediating protective effects in the retina.


Asunto(s)
Anemia/metabolismo , Eritropoyetina/metabolismo , Fallo Renal Crónico/metabolismo , Hígado/enzimología , Prolil Hidroxilasas/deficiencia , Enfermedades de la Retina/metabolismo , Anemia/patología , Animales , Western Blotting , Modelos Animales de Enfermedad , Fallo Renal Crónico/patología , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Enfermedades de la Retina/patología
9.
Am J Pathol ; 184(3): 686-96, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24440788

RESUMEN

Deficiencies in prolyl hydroxylase domain proteins (PHDs) may lead to the accumulation of hypoxia-inducible factor-α proteins, the latter of which activate local angiogenic responses by paracrine mechanisms. Here, we investigate whether a keratinocyte-specific PHD deficiency may promote vascular survival and growth in a distantly located ischemic tissue by a remote signaling mechanism. We generated mice that carry a keratinocyte-specific Phd2 knockout (kPhd2KO) and performed femoral artery ligation. Relative to wild-type controls, kPhd2KO mice displayed improved vascular survival and arteriogenesis in ischemic hind limbs, leading to the accelerated recovery of hindlimb perfusion and superior muscle regeneration. Similar protective effects were also seen in type 1 and type 2 diabetic mice. Molecularly, both abundance of hypoxia-inducible factor-1α protein and expression of vascular endothelial growth factor-A were increased in epidermal tissues of kPhd2KO mice, accompanied by increased plasma concentration of vascular endothelial growth factor-A. Contrary to kPhd2KO mice, which are PHD2 deficient in all skin tissues, localized kPhd2KO in hindlimb skin tissues did not have similar effects, excluding paracrine signaling as a major mechanism. Confirming the existence of remote effects, hepatocyte-specific Phd2 knockout also protected hind limbs from ischemia injury. These data indicate that vascular survival and growth in ischemia-injured tissue may be stimulated by suppressing PHD2 in a remotely located tissue and may provide highly effective angiogenesis therapies without the need for directly accessing target tissues.


Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Miembro Posterior/irrigación sanguínea , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Isquemia/fisiopatología , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Arteria Femoral/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Masculino , Ratones , Ratones Noqueados , Factor A de Crecimiento Endotelial Vascular/metabolismo
10.
Blood ; 121(8): 1436-45, 2013 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-23264599

RESUMEN

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2α. In contrast, HIF-1α served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1α resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2α-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2α-dependent erythrocytosis, whereas HIF-1α protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hematopoyesis Extramedular/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Policitemia/genética , Procolágeno-Prolina Dioxigenasa/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Encéfalo/fisiología , Células Cultivadas , Eritropoyetina/genética , Eritropoyetina/metabolismo , Femenino , Fibroblastos/citología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Queratinocitos/citología , Riñón/citología , Riñón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Policitemia/metabolismo , Policitemia/patología , Procolágeno-Prolina Dioxigenasa/metabolismo , Índice de Severidad de la Enfermedad , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patología
11.
Microvasc Res ; 97: 181-8, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25446011

RESUMEN

BACKGROUND: There is an emerging focus on investigating innovative therapeutic molecules that can potentially augment neovascularization in order to treat peripheral arterial disease (PAD). Although prolyl hydroxylase domain proteins 1 and 3 (PHD1 and PHD3) may modulate angiogenesis via regulation of hypoxia inducible factor-1α (HIF-1α), there has been no study directly addressing their roles in ischemia-induced vascular growth. We hypothesize that PHD1(-/-) or PHD3(-/-) deficiency might promote angiogenesis in the murine hind-limb ischemia (HLI) model. STUDY DESIGN: Wild type (WT), PHD1(-/-) and PHD3(-/-) male mice aged 8-12weeks underwent right femoral artery ligation. Post-procedurally, motor function assessment and laser Doppler imaging were periodically performed. The mice were euthanized after 28days and muscles were harvested. Immunohistochemical analysis was performed to determine the extent of angiogenesis by measuring capillary and arteriolar density. VEGF expression was quantified by enzyme-linked immunosorbent assay (ELISA). Bcl-2 and HIF-1α were analyzed by immunofluorescence. Fibrosis was measured by picrosirius red staining. RESULTS: PHD1(-/-) and PHD3(-/-) mice showed significantly improved recovery of perfusion and motor function score when compared to WT after femoral artery ligation. These mice also exhibited increased capillary and arteriolar density, capillary/myocyte ratio along with decreased fibrosis compared to WT. VEGF, Bcl-2 and HIF-1α expression increased in PHD1(-/-) and PHD3(-/-) mice compared to WT. CONCLUSIONS: Taken together these results suggest that PHD1 and PHD3 deletions promote angiogenesis in ischemia-injured tissue, and may present a promising therapeutic strategy in treating PAD.


Asunto(s)
Eliminación de Gen , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Neovascularización Fisiológica , Procolágeno-Prolina Dioxigenasa/deficiencia , Animales , Modelos Animales de Enfermedad , Fibrosis , Miembro Posterior , Isquemia/genética , Isquemia/patología , Isquemia/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Músculo Esquelético/patología , Procolágeno-Prolina Dioxigenasa/genética , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Recuperación de la Función , Flujo Sanguíneo Regional , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismo
12.
Stem Cells ; 32(6): 1564-77, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24307555

RESUMEN

CD13 is a multifunctional cell surface molecule that regulates inflammatory and angiogenic mechanisms in vitro, but its contribution to these processes in vivo or potential roles in stem cell biology remains unexplored. We investigated the impact of loss of CD13 on a model of ischemic skeletal muscle injury that involves angiogenesis, inflammation, and stem cell mobilization. Consistent with its role as an inflammatory adhesion molecule, lack of CD13 altered myeloid trafficking in the injured muscle, resulting in cytokine profiles skewed toward a prohealing environment. Despite this healing-favorable context, CD13(KO) animals showed significantly impaired limb perfusion with increased necrosis, fibrosis, and lipid accumulation. Capillary density was correspondingly decreased, implicating CD13 in skeletal muscle angiogenesis. The number of CD45-/Sca1-/α7-integrin+/ß1-integrin+ satellite cells was markedly diminished in injured CD13(KO) muscles and adhesion of isolated CD13(KO) satellite cells was impaired while their differentiation was accelerated. Bone marrow transplantation studies showed contributions from both host and donor cells to wound healing. Importantly, CD13 was coexpressed with Pax7 on isolated muscle-resident satellite cells. Finally, phosphorylated-focal adhesion kinase and ERK levels were reduced in injured CD13(KO) muscles, consistent with CD13 regulating satellite cell adhesion, potentially contributing to the maintenance and renewal of the satellite stem cell pool and facilitating skeletal muscle regeneration.


Asunto(s)
Antígenos CD13/metabolismo , Diferenciación Celular , Isquemia/metabolismo , Isquemia/patología , Células Satélite del Músculo Esquelético/patología , Células Madre/patología , Animales , Arteriopatías Oclusivas/metabolismo , Arteriopatías Oclusivas/patología , Arteriopatías Oclusivas/fisiopatología , Arterias/metabolismo , Arterias/patología , Adhesión Celular , Recuento de Células , Citocinas/metabolismo , Inflamación/patología , Isquemia/fisiopatología , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Recuperación de la Función , Regeneración , Transducción de Señal , Células Madre/metabolismo , Cicatrización de Heridas
13.
Curr Atheroscler Rep ; 17(6): 510, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25876920

RESUMEN

Injury of arterial endothelium by abnormal shear stress and other insults induces migration and proliferation of vascular smooth muscle cells (VSMCs), which in turn leads to intimal thickening, hypoxia, and vasa vasorum angiogenesis. The resultant new blood vessels extend from the tunica media into the outer intima, allowing blood-borne oxidized low-density lipoprotein (oxLDL) particles to accumulate in outer intimal tissues by extravasation through local capillaries. In response to oxLDL accumulation, monocytes infiltrate into arterial wall tissues, where they differentiate into macrophages and subsequently evolve into foam cells by uptaking large quantities of oxLDL particles, the latter process being stimulated by hypoxia. Increased oxygen demand due to expanding macrophage and foam cell populations contributes to persistent hypoxia in plaque lesions, whereas hypoxia further promotes plaque growth by stimulating angiogenesis, monocyte infiltration, and oxLDL uptake into macrophages. Molecularly, the accumulation of hypoxia-inducible factor (HIF)-1α and the expression of its target genes mediate many of the hypoxia-induced processes during plaque initiation and growth. It is hoped that further understanding of the underlying mechanisms may lead to novel therapies for effective intervention of atherosclerosis.


Asunto(s)
Aterosclerosis/metabolismo , Endotelio Vascular/metabolismo , Células Espumosas/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Endotelio Vascular/patología , Humanos , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/patología
14.
Circ Res ; 112(8): 1135-49, 2013 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-23476056

RESUMEN

RATIONALE: NADPH oxidase (Nox) 2 and Nox4 are major components of the Nox family which purposefully produce reactive oxidative species, namely O2(-) and H2O2, in the heart. The isoform-specific contribution of Nox2 and Nox4 to ischemia/reperfusion (I/R) injury is poorly understood. OBJECTIVE: We investigated the role of Nox2 and Nox4 in mediating oxidative stress and myocardial injury during I/R using loss-of-function mouse models. METHODS AND RESULTS: Systemic (s) Nox2 knockout (KO), sNox4 KO, and cardiac-specific (c) Nox4 KO mice were subjected to I/R (30 minutes/24 hours, respectively). Both myocardial infarct size/area at risk and O2(-) production were lower in sNox2 KO, sNox4 KO, and cNox4 KO than in wild-type mice. Unexpectedly, however, the myocardial infarct size/area at risk was greater, despite less O2(-) production, in sNox2 KO+cNox4 KO (double-KO) mice and transgenic mice (Tg) with cardiac-specific expression of dominant-negative Nox, which suppresses both Nox2 and Nox4, than in wild-type or single KO mice. Hypoxia-inducible factor-1α was downregulated whereas peroxisome proliferator-activated receptor-α was upregulated in Tg-dominant-negative Nox mice. A cross with mice deficient in prolyl hydroxylase 2, which hydroxylates hypoxia-inducible factor-1α, rescued the I/R injury and prevented upregulation of peroxisome proliferator-activated receptor-α in Tg-dominant-negative Nox mice. A cross with peroxisome proliferator-activated receptor-α KO mice also attenuated the injury in Tg- dominant-negative Nox mice. CONCLUSIONS: Both Nox2 and Nox4 contribute to the increase in reactive oxidative species and injury by I/R. However, low levels of reactive oxidative species produced by either Nox2 or Nox4 regulate hypoxia-inducible factor-1α and peroxisome proliferator-activated receptor-α, thereby protecting the heart against I/R, suggesting that Noxs also act as a physiological sensor for myocardial adaptation.


Asunto(s)
Regulación hacia Abajo/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Glicoproteínas de Membrana/deficiencia , NADPH Oxidasas/deficiencia , PPAR alfa/biosíntesis , Daño por Reperfusión/metabolismo , Regulación hacia Arriba/fisiología , Animales , Activación Enzimática/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , PPAR alfa/fisiología , Daño por Reperfusión/enzimología , Daño por Reperfusión/fisiopatología
15.
Circulation ; 127(21): 2078-87, 2013 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-23630130

RESUMEN

BACKGROUND: Recent studies suggest that the oxygen-sensing pathway consisting of transcription factor hypoxia-inducible factor and prolyl hydroxylase domain proteins (PHDs) plays a critical role in glucose metabolism. However, the role of adipocyte PHD in the development of obesity has not been clarified. We examined whether deletion of PHD2, the main oxygen sensor, in adipocytes affects diet-induced obesity and associated metabolic abnormalities. METHODS AND RESULTS: To delete PHD2 in adipocyte, PHD2-floxed mice were crossed with aP2-Cre transgenic mice (Phd2(f/f)/aP2-Cre). Phd2(f/f)/aP2-Cre mice were resistant to high-fat diet-induced obesity (36.7±1.7 versus 44.3±2.0 g in control; P<0.01) and showed better glucose tolerance and homeostasis model assessment-insulin resistance index than control mice (3.6±1.0 versus 11.1±2.1; P<0.01). The weight of white adipose tissue was lighter (epididymal fat, 758±35 versus 1208±507 mg in control; P<0.01) with a reduction in adipocyte size. Macrophage infiltration into white adipose tissue was also alleviated in Phd2(f/f)/aP2-Cre mice. Target genes of hypoxia-inducible factor, including glycolytic enzymes and adiponectin, were upregulated in adipocytes of Phd2(f/f)/aP2-Cre mice. Lipid content was decreased and uncoupling protein-1 expression was increased in brown adipose tissue of Phd2(f/f)/aP2-Cre mice. Knockdown of PHD2 in 3T3L1 adipocytes induced a decrease in the glucose level and an increase in the lactate level in the supernatant with upregulation of glycolytic enzymes and reduced lipid accumulation. CONCLUSIONS: PHD2 in adipose tissue plays a critical role in the development of diet-induced obesity and glucose intolerance. PHD2 might be a novel target molecule for the treatment of obesity and associated metabolic abnormalities.


Asunto(s)
Dieta Alta en Grasa/efectos adversos , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/fisiopatología , Obesidad/etiología , Obesidad/fisiopatología , Procolágeno-Prolina Dioxigenasa/fisiología , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo Blanco/irrigación sanguínea , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Transportador de Glucosa de Tipo 4/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Metabolismo de los Lípidos/fisiología , Macrófagos/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/metabolismo , Neovascularización Fisiológica/fisiología , Obesidad/patología , Consumo de Oxígeno/fisiología , Procolágeno-Prolina Dioxigenasa/deficiencia , Procolágeno-Prolina Dioxigenasa/genética
16.
Circulation ; 126(6): 741-52, 2012 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-22753193

RESUMEN

BACKGROUND: Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. Whereas germline Vegfr-1(-/-) embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain appear healthy. METHODS AND RESULTS: We performed Cre-loxP-mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell proliferation and facilitated angiogenesis of blood vessels that matured and perfused properly. Vascular permeability was normal at the basal level but elevated in response to high doses of exogenous VEGF-A. In the postinfarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of the VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2(+/-) heterozygosity into Vegfr-1 somatic knockout mice. CONCLUSIONS: Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1-deficient mice.


Asunto(s)
Neovascularización Fisiológica/genética , Regulación hacia Arriba , Receptor 1 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología
17.
Biol Open ; 12(1)2023 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-36625299

RESUMEN

Tailless (TLX, an orphan nuclear receptor) and hypoxia inducible factor-2α (HIF2α) are both essential for retinal astrocyte and vascular development. Tlx-/- mutation and astrocyte specific Hif2α disruption in Hif2αf/f/GFAPCre mice are known to cause defective astrocyte development and block vascular development in neonatal retinas. Here we report that TLX and HIF2α support retinal angiogenesis by cooperatively maintaining retinal astrocytes in their proangiogenic states. While Tlx+/- and Hif2αf/+/GFAPCre mice are phenotypically normal, Tlx+/-/Hif2αf/+/GFAPCre mice display precocious retinal astrocyte differentiation towards non-angiogenic states, along with significantly reduced retinal angiogenesis. In wild-type mice, TLX and HIF2α coexist in the same protein complex, suggesting a cooperative function under physiological conditions. Furthermore, astrocyte specific disruption of Phd2 (prolyl hydroxylase domain protein 2), a manipulation previously shown to cause HIF2α accumulation, did not rescue retinal angiogenesis in Tlx-/- background, which suggests functional dependence of HIF2α on TLX. Finally, the expression of fibronectin and VEGF-A is significantly reduced in retinal astrocytes of neonatal Tlx+/-/Hif2αf/+/GFAPCre mice. Overall, these data indicate that TLX and HIF2α cooperatively support retinal angiogenesis by maintaining angiogenic potential of retinal astrocytes.


Asunto(s)
Astrocitos , Neuroglía , Animales , Ratones , Astrocitos/metabolismo , Animales Recién Nacidos , Retina/metabolismo , Hipoxia/metabolismo
18.
Nat Cell Biol ; 25(7): 950-962, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37400498

RESUMEN

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.


Asunto(s)
Necroptosis , Receptores Tipo I de Factores de Necrosis Tumoral , Humanos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Hidroxilación , Hipoxia , Prolina/metabolismo , Inflamación , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo
19.
Hum Mol Genet ; 19(21): 4145-59, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20705734

RESUMEN

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene coding for the protein dystrophin. Recent work demonstrates that dystrophin is also found in the vasculature and its absence results in vascular deficiency and abnormal blood flow. This induces a state of ischemia further aggravating the muscular dystrophy pathogenesis. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship between muscular dystrophy and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor, and therefore both homozygous (Flt-1(-/-)) and heterozygous (Flt-1(+/-)) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during embryogenesis. Here, we show that Flt-1(+/-) and mdx:Flt-1(+/-) adult mice also display a developmentally increased vascular density in skeletal muscle compared with the wild-type and mdx mice, respectively. The mdx:Flt-1(+/-) mice show improved muscle histology compared with the mdx mice with decreased fibrosis, calcification and membrane permeability. Functionally, the mdx:Flt-1(+/-) mice have an increase in muscle blood flow and force production, compared with the mdx mice. Consequently, the mdx:utrophin(-/-):Flt-1(+/-) mice display improved muscle histology and significantly higher survival rates compared with the mdx:utrophin(-/-) mice, which show more severe muscle phenotypes than the mdx mice. These data suggest that increasing the vasculature in DMD may ameliorate the histological and functional phenotypes associated with this disease.


Asunto(s)
Haploinsuficiencia , Distrofia Muscular Animal/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Proliferación Celular , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Contracción Muscular , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Fenotipo
20.
Am J Pathol ; 178(4): 1881-90, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21435465

RESUMEN

Retinopathy of prematurity is a major side effect of oxygen therapy for preterm infants, and is a leading cause of blindness in children. To date, it remains unclear whether the initial microvascular obliteration is triggered by degradation of hypoxia inducible factor (HIF) α proteins or by other mechanisms such as oxidative stress. Here we show that prolyl hydroxylase domain protein 2 (PHD2), an enzyme mostly responsible for oxygen-induced degradation of HIF-α proteins, plays a major role in oxygen-induced retinopathy in mice. In neonatal mice expressing normal amounts of PHD2, exposure to 75% oxygen caused significant degradation of retinal HIF-α proteins, accompanied by massive losses of retinal microvessels. PHD2 deficiency significantly stabilized HIF-1α, and to some extent HIF-2α, in neonatal retinal tissues, and protected retinal microvessels from oxygen-induced obliteration. After hyperoxia-treated neonatal mice were returned to ambient room air, retinal vasculature in PHD2-deficient mice remained mostly intact and showed very little neoangiogenesis. These findings demonstrate a close association between PHD2-dependent HIF-α degradation and oxygen-induced retinal microvascular obliteration, and imply that PHD2 may be a promising therapeutic target to prevent oxygen-induced retinopathy.


Asunto(s)
Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/fisiología , Enfermedades de la Retina/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Hiperoxia/metabolismo , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Inmunohistoquímica/métodos , Ratones , Ratones Noqueados , Pericitos/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Estructura Terciaria de Proteína , Vasos Retinianos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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