RESUMEN
BACKGROUND: Diabetes mellitus is an important cardiovascular risk factor characterized by elevated plasma glucose levels. High glucose (HG) negatively influences endothelial cell (EC) function, which is characterized by the inability of ECs to respond to vascular endothelial growth factor (VEGF-A) stimulation. We aimed to identify potential strategies to improve EC function in diabetes. METHODS AND RESULTS: Human umbilical cord endothelial cells (HUVECs) were subjected to hyperglycemic milieu by exposing cells to HG together with glucose metabolite, methylglyoxal (MG) in vitro. Hyperglycemic cells showed reduced chemotactic responses towards VEGF-A as revealed by Boyden chamber migration assays, indicating the development of "VEGF resistance" phenotype. Furthermore, HG/MG-exposed cells were defective in their general migratory and proliferative responses and were in a pro-apoptotic state. Mechanistically, the exposure to HG/MG resulted in reactive oxygen species (ROS) accumulation which is secondary to the impairment of thioredoxin (Trx) activity in these cells. Pharmacological and genetic targeting of Trx recapitulated VEGF resistance. Functional supplementation of Trx using thioredoxin mimetic peptides (TMP) reversed the HG/MG-induced ROS generation, improved the migration, proliferation, survival and restored VEGF-A-induced chemotaxis and sprouting angiogenesis of hyperglycemic ECs. Importantly, TMP treatment reduced ROS accumulation and improved VEGF-A responses of placental arterial endothelial cells isolated from gestational diabetes mellitus patients. CONCLUSIONS: Our findings suggest a putative role for Trx in modulating EC function and its functional impairment in HG conditions contribute to EC dysfunction. Supplementation of TMP could be used as a novel strategy to improve endothelial cell function in diabetes.
Asunto(s)
Hiperglucemia , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Supervivencia Celular , Células Cultivadas , Células Endoteliales , Femenino , Humanos , Hiperglucemia/tratamiento farmacológico , Embarazo , Tiorredoxinas , Factor A de Crecimiento Endotelial VascularRESUMEN
Diabetes mellitus (DM) is a major cardiovascular risk factor contributing to cardiovascular complications by inducing vascular cell dysfunction. Monocyte dysfunction could contribute to impaired arteriogenesis response in DM patients. DM monocytes show blunted chemotactic responses to arteriogenic stimuli, a condition termed as vascular endothelial growth factor (VEGF) resistance. We hypothesize that methylglyoxal (MG), a glucose metabolite, induces monocyte dysfunction and aimed to elucidate the underlying molecular mechanisms. Human monocytes exposed to MG or monocytes from DM patients or mice (db/db) showed VEGF-resistance secondary to a pro-migratory phenotype. Mechanistically, DM conditions or MG exposure resulted in the upregulation of the expression of SHP-2 phosphatase. This led to the enhanced activity of SHP-2 and aided an interaction with SRC kinase. SHP-2 dephosphorylated the inhibitory phosphorylation site of SRC leading to its abnormal activation and phosphorylation of cytoskeletal protein, paxillin. We demonstrated that MG-induced molecular changes could be reversed by pharmacological inhibitors of SHP-2 and SRC and by genetic depletion of SHP-2. Finally, a SHP-2 inhibitor completely reversed the dysfunction of monocytes isolated from DM patients and db/db mice. In conclusion, we identified SHP-2 as a hitherto unknown target for improving monocyte function in diabetes. This opens novel perspectives for treating diabetic complications associated with impaired monocyte function.
Asunto(s)
Hiperglucemia/patología , Monocitos/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Piruvaldehído/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Familia-src Quinasas/metabolismo , Animales , Quimiotaxis , Humanos , Ratones , Monocitos/efectos de los fármacosRESUMEN
In the current paradigm on coronary collateral development, it is assumed that these vessels develop consequentially from increased fluid shear stress (FSS) through preexisting collateral arteries. The increased FSS follows from an increase in pressure gradient between the region at risk and well-perfused surroundings. The objective of this study was to test the hypothesis that, in the heart, collateral connections can form in the absence of an increased FFS and consequentially at any depth and region within the ventricular wall. In Yorkshire pigs, gradual left circumflex coronary artery occlusion was obtained over 6 wk by an ameroid constrictor, whereas the control group underwent a sham operation. Hearts were harvested and subsequently processed in an imaging cryomicrotome, resulting in 40-µm voxel resolution three-dimensional reconstructions of the intramural vascular vessels. Dedicated software segmented the intramural vessels and all continuous vascular pathways containing a collateral connection. In the ameroid group, 192 collaterals, 22-1,049 µm in diameter, were detected with 62% within the subendocardium. Sixty percent of collaterals bridged from the left anterior descending artery to left circumflex coronary artery. A novel result is that 25% (n = 48) of smaller-radius collaterals (P = 0.047) connected with both origin and terminus in the nontarget area where perfusion was assumed uncompromised. In the porcine heart, collateral vessels develop not only in ischemic border zones with increased FSS but also away from such border zones where increased FSS is unlikely. The majority of collaterals were located at the subendocardium, corresponding to the region with highest prevalence for ischemia.
Asunto(s)
Presión Sanguínea/fisiología , Circulación Colateral/fisiología , Vasos Coronarios/crecimiento & desarrollo , Isquemia Miocárdica/fisiopatología , Neovascularización Fisiológica/fisiología , Algoritmos , Animales , Vasos Coronarios/fisiología , Masculino , Modelos Animales , PorcinosRESUMEN
OBJECTIVE: Notch has been implicated in neointima formation as reflected by increased Notch/Jagged expression on vascular injury and the promigratory effect of Notch signaling on smooth muscle cells. Soluble Jagged-1 (sJag1) has been shown to inhibit Notch signaling in vitro; however, its capacity to suppress neointima formation remains unknown. METHODS AND RESULTS: Balloon injury of rat carotid arteries induced Notch1, Notch3, and Jagged-1 expression at days 3 and 14 postinjury. Notch signaling was activated as shown by increased expression of the Notch target gene Herp2. Adenoviral sJag1 (Ad-sJag1) transfection reduced neointima formation in carotid artery and enhanced reendothelialization, whereas adenoviral full-length Jagged-1 (Ad-Fl-Jag1) or LacZ had no effect. Injury-induced Herp2 expression was absent in vessels treated with Ad-sJag1. Consistently, Herp2 expression was reduced in Ad-sJag1-infected or recombinant sJag1 -treated coronary artery smooth muscle cells (CASMCs). Ad-sJag1 had no effect on human umbilical endothelial cell behavior, but it significantly reduced proliferation and migration of CASMCs. Overexpression of Herp2 in sJag1-treated CASMCs rescued the migratory and proliferative capacity in vitro. CONCLUSIONS: Our results demonstrate that sJag1 can inhibit neointima formation after balloon injury by decreasing smooth muscle cell proliferation and migration through interference with Notch-Herp2 signaling.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/prevención & control , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Receptores Notch/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Túnica Íntima/metabolismo , Análisis de Varianza , Animales , Proteínas de Unión al Calcio/genética , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Hiperplasia , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Ratas , Ratas Sprague-Dawley , Receptor Notch3 , Proteínas Serrate-Jagged , Factores de Tiempo , Transfección , Túnica Íntima/patologíaRESUMEN
The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.
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Comunicación Celular/fisiología , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Receptores Notch/metabolismo , Elementos de Respuesta/fisiología , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de Unión al Calcio , Células Cultivadas , Vasos Coronarios/citología , Células Endoteliales/citología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Estructura Terciaria de Proteína , Receptores Notch/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Therapeutic neovascularization for cardiovascular ischemia is a promising avenue in spite of disappointing early clinical trial results. The concept of three different mechanisms of neovascularization has served to define potential therapeutic targets such as vascular remodeling and stem cell recruitment, but it is anticipated that this will lose significance as the pleiotropic nature of angiogenic cytokines becomes fully understood. With the rapidly growing body of data on growth factors and pro-angiogenic strategies, approaches will emerge that are more effective than the ones that have been tested clinically thus far. Combinations of growth factors, for instance to stabilize vessels, or growth factors combined with cell transplants deserve more attention but will make the design of preclinical and clinical studies increasingly complex. Recent developments suggest that when using the appropriate dose and treatment regimens, even single growth factor therapy can result in stable and functional vessels. Whether gene therapy or protein therapy will be optimal for this purpose depends mainly on technical developments in vector design and production and on progress in the engineering of slow release matrix formulations for proteins. With the increasing complexity of therapeutic strategies, it remains imperative that these approaches are rationally based on fundamental and preclinical data.