RESUMEN
Periods of low energy supply are challenging conditions for organisms and cells during fasting or famine. Although changes in nutrient levels in the blood are first sensed by endothelial cells, studies on their metabolic adaptations to diminished energy supply are lacking. We analyzed the dynamic metabolic activity of human umbilical vein endothelial cells (HUVECs) in basal conditions and after serum starvation. Metabolites of glycolysis, the tricarboxylic acid (TCA) cycle, and the glycerol pathway showed lower levels after serum starvation, whereas amino acids had increased levels. A metabolic flux analysis with 13C-glucose or 13C-glutamine labeling for different time points reached a plateau phase of incorporation after 30 h for 13C-glucose and after 8 h for 13C-glutamine under both experimental conditions. Notably, we observed a faster label incorporation for both 13C-glucose and 13C-glutamine after serum starvation. In the linear range of label incorporation after 3 h, we found a significantly faster incorporation of central carbon metabolites after serum starvation compared to the basal state. These findings may indicate that endothelial cells develop increased metabolic activity to cope with energy deficiency. Physiologically, it can be a prerequisite for endothelial cells to form new blood vessels under unfavorable conditions during the process of angiogenesis in vivo.
Asunto(s)
Glutamina , Inanición , Humanos , Glutamina/metabolismo , Aminoácidos/metabolismo , Glucólisis , Glucosa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismoRESUMEN
Hyperplasia and hypertrophy of fat cells can be found in obesity and increased adiposity is associated with endothelial dysfunction as an early event of atherosclerosis. However, it is unclear whether human adipocytes directly influence endothelial protein secretion. To study the crosstalk between fat and endothelial cells, human umbilical venous endothelial cells (HUVECs) were cultured in infranatants (Adipo) of primary differentiated human adipocytes. Interestingly, significantly increased secretion of 23 cytokines and chemokines from HUVECs was detected in four independent experiments after Adipo stimulation by protein array analysis detecting a total of 174 different proteins. Among those, time-dependent Adipo-induced upregulation of cytokine secretion in HUVECs was confirmed by ELISA for interleukin (IL)-8, monokine induced by gamma interferon, macrophage inflammatory protein (MIP)-1beta, MIP-3alpha, monocyte chemoattractant protein-1, and IL-6. Factors besides adiponectin, leptin, resistin, and tumor necrosis factor alpha appear to mediate these stimulatory effects. Our findings suggest that endothelial cell secretion is significantly influenced towards a proinflammatory pattern by adipocyte-secreted factors.
Asunto(s)
Adipocitos/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Comunicación Paracrina/inmunología , Adipocitos/inmunología , Células Cultivadas , Células Endoteliales/inmunología , Endotelio Vascular/citología , Humanos , Mediadores de Inflamación , Proteínas/análisis , Proteínas/metabolismo , Proteómica , Regulación hacia ArribaRESUMEN
BACKGROUND AND AIMS: Gonadal hormones are mainly thought to account for sex and gender differences in the incidence, clinical manifestation and therapy of many cardiovascular diseases. However, intrinsic sex differences at the cellular level are mostly overlooked. Here, we assessed sex-specific metabolic and functional differences between male and female human umbilical vein endothelial cells (HUVECs). METHODS: Cellular metabolism was investigated by bioenergetic studies (Seahorse Analyser) and a metabolomic approach. Protein levels were determined by Western blots and proteome analysis. Vascular endothelial growth factor (VEGF)-stimulated cellular migration was assessed by gap closure. HUVECs from dizygotic twin pairs were used for most experiments. RESULTS: No sex differences were observed in untreated cells. However, sexual dimorphisms appeared after stressing the cells by serum starvation and treatment with VEGF. Under both conditions, female cells had higher intracellular ATP and metabolite levels. A significant decline in ATP levels was observed in male cells after serum starvation. After VEGF, the ratio of glycolysis/mitochondrial respiration was higher in female cells and migration was more pronounced. CONCLUSIONS: These results point to an increased stress tolerance of female cells. We therefore propose that female cells have an energetic advantage over male cells under conditions of diminished nutrient supply. A more favourable energy balance of female HUVECs after serum starvation and VEGF could potentially explain their stronger migratory capacity.
Asunto(s)
Movimiento Celular , Metabolismo Energético , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica , Gemelos Dicigóticos , Inductores de la Angiogénesis/farmacología , Movimiento Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/metabolismo , Metabolismo Energético/efectos de los fármacos , Femenino , Humanos , Masculino , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Mapas de Interacción de Proteínas , Caracteres Sexuales , Factores Sexuales , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Endothelial nitric oxide synthase (eNOS) is the key enzyme of vascular homeostasis involved in the pathophysiology of several cardiovascular diseases. The aim of our study was to investigate whether eNOS expression and activity are regulated by the proteasome. Bovine pulmonary artery endothelial cells (CPAE cells) were treated with the proteasome inhibitor MG132. MG132 (50-250 nmol/L) dose-dependently increased mRNA and protein levels of eNOS. Comparable results were obtained with other specific proteasome inhibitors, whereas the nonproteasomal calpain and cathepsin inhibitor ALLM had no effect. Efficacy of proteasome inhibition was evidenced by accumulation of poly-ubiquitinylated proteins and by measuring proteasomal activity in cell extracts. Cycloheximide prevented up-regulation of eNOS protein, indicating that post-translational stabilization of eNOS is not involved. eNOS activity was increased up to 2.8-fold (MG132 100 nmol/L, 48 h). Incubation of rat aortic rings with MG132 significantly enhanced endothelial-dependent vasorelaxation. Single MG132 treatment (100 nmol/L) induced long-term effects in CPAE cells, with increases of eNOS protein and activity for up to 10 days. Our results indicate that low-dose proteasome inhibition enhances eNOS expression and activity, and improves endothelial function.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Leupeptinas/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Ubiquitina/antagonistas & inhibidores , Ubiquitina/metabolismo , Animales , Bovinos , Cisteína Endopeptidasas/metabolismo , Endotelio Vascular/enzimología , Endotelio Vascular/fisiología , Masculino , Complejos Multienzimáticos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo III , Complejo de la Endopetidasa Proteasomal , Arteria Pulmonar , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , VasodilataciónRESUMEN
Hyperplasia and hypertrophy of fat cells can be found in obesity, and increased adiposity is associated with endothelial dysfunction as an early event of atherosclerosis. However, it is unclear whether human adipocytes directly influence endothelial function. To study the crosstalk between fat and endothelial cells, human umbilical venous endothelial cells (HUVECs), and human coronary artery endothelial cells (HCAECs) were cultured in infranatants (Adipo) of primary differentiated human adipocytes. Interestingly, incubation of HUVECs and HCAECs with Adipo significantly increased monocyte adhesion 7.3 and 2.2-fold, respectively. VCAM-1, ICAM-1, and E-selectin in HUVECs were upregulated 3.9, 3.0, and 9.5-fold, respectively, under these conditions. Furthermore, Adipo significantly stimulated NFkappaB activity 1.9-fold. The NFkappaB inhibitor MG-132 and heat inactivation significantly reversed Adipo-stimulated monocyte adhesion. TNFalpha-neutralizing antibodies partly reversed Adipo-induced monocyte adhesion. In contrast, thiazolidinedione-pretreatment of human adipocytes did not alter the effects of Adipo. Adipo did not show cytotoxic effects. Taken together, we demonstrate that endothelial dysfunction is induced by adipocyte-secreted factors via NFkappaB partly dependent on TNFalpha.