Vascular dysfunction that accompanies obesity and insulin resistance may be mediated by lipid metabolites. We sought to determine if vascular ceramide leads to arterial dysfunction and to elucidate the underlying mechanisms. Pharmacological inhibition of de novo ceramide synthesis, using the Ser palmitoyl transferase inhibitor myriocin, and heterozygous deletion of dihydroceramide desaturase prevented vascular dysfunction and hypertension in mice after high-fat feeding. These findings were recapitulated in isolated arteries in vitro, confirming that ceramide impairs endothelium-dependent vasorelaxation in a tissue-autonomous manner. Studies in endothelial cells reveal that de novo ceramide biosynthesis induced protein phosphatase 2A (PP2A) association directly with the endothelial nitric oxide synthase (eNOS)/Akt/Hsp90 complex that was concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and by dephosphorylating eNOS. Ceramide decreased the association between PP2A and the predominantly cytosolic inhibitor 2 of PP2A. We conclude that ceramide mediates obesity-related vascular dysfunction by a mechanism that involves PP2A-mediated disruption of the eNOS/Akt/Hsp90 signaling complex. These results provide important insight into a pathway that represents a novel target for reversing obesity-related vascular dysfunction.
Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance.
The mechanisms of sensory transduction of pulmonary reactive oxygen species (ROS) by capsaicin-sensitive vagal lung afferent fibres are unclear. To investigate the role of transient receptor potential vanilloid 1 (TRPV1) receptors and P2X purinoceptors in this sensory transduction, we recorded fibre activity (FA) from 132 fibres of this type in 132 anaesthetized and ventilated rats. Airway challenge of aerosolized H 2 O 2 (0, 0.2 and 0.4%) produced a concentration-dependant fibre stimulation. The fibre responses to 0.4% H 2 O 2 were attenuated by dimethylthiourea (a hydroxyl radical (·OH) scavenger; change in fibre activity (∆FA), −55 ± 9%) or deferoxamine (an iron-chelator that prevents formation of ·OH; ∆FA, −59 ± 9%), were prevented by catalase (an enzyme catalysing H 2 O 2 ; ∆FA, −96 ± 3%) and were unaffected by the vehicle for dimethylthiourea, iron-saturated deferoxamine or heat-inactivated catalase. The fibre responses to 0.4% H 2 O 2 were attenuated by capsazepine (a TRPV1 receptor antagonist; ∆FA, −39 ± 9%) or iso-pyridoxalphosphate-6-azophenyl-2 ,5 -disulphonate (iso-PPADS, a P2X receptor antagonist; ∆FA, −51 ± 9%), were further reduced by capsazepine and iso-PPADS in combination (∆FA, −70 ± 13%), and were unaltered by their vehicles. The fibre responses to cigarette smoke (20 ml), an irritant that generates ROS, were attenuated by dimethylthiourea (∆FA, −61 ± 9%) or capsazepine and iso-PPADS in combination (∆FA, −67 ± 9%). These results suggest that both the TRPV1 and P2X receptors mediate the sensory transduction of ROS, especially H 2 O 2 and ·OH, by capsaicin-sensitive vagal lung afferent fibres.
Autophagy is a lysosomal catabolic process by which cells degrade or recycle their contents to maintain cellular homeostasis, adapt to stress, and respond to disease. Impairment of autophagy in endothelial cells studied under static conditions results in oxidant stress and impaired nitric oxide (NO) bioavailability. We tested the hypothesis that vascular autophagy is also important for induction of NO production caused by exposure of endothelial cells to shear stress (i.e., 3 h × ≈20 dyn/cm(2)). Atg3 is a requisite autophagy pathway mediator. Control cells treated with non-targeting control siRNA showed increased autophagy, reactive oxygen species (ROS) production, endothelial NO synthase (eNOS) phosphorylation, and NO production upon exposure to shear stress (p < 0.05 for all). In contrast, cells with >85% knockdown of Atg3 protein expression (via Atg3 siRNA) exhibited a profound impairment of eNOS phosphorylation, and were incapable of increasing NO in response to shear stress. Moreover, ROS accumulation and inflammatory cytokine production (MCP-1 and IL-8) were exaggerated (all p < 0.05) in response to shear stress. These findings reveal that autophagy not only plays a critical role in maintaining NO bioavailability, but may also be a key regulator of oxidant-antioxidant balance and inflammatory-anti-inflammatory balance that ultimately regulate endothelial cell responses to shear stress.
Objective Impaired endothelial cell autophagy compromises shear-stress induced nitric oxide (NO) generation. We determined the responsible mechanism. Approach and Results Upon autophagy compromise in bovine aortic endothelial cells (ECs) exposed to shear-stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y-1 receptors, secondary to impaired autophagy in ECs, prevents shear-induced p-eNOSS1177 and NO generation. Maneuvers that restore glucose transport and glycolysis (e.g., overexpression of GLUT1) or purinergic signaling (e.g., addition of exogenous ADP) rescue shear-induced p-eNOSS1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose-transport via GLUT1 siRNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (e.g., apyrase), or inhibiting P2Y1 receptors using pharmacological (e.g., MRS2179) or genetic (e.g., P2Y1-R siRNA) procedures, inhibits shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKCδT505 in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that: (i) shear-stress induced activating phosphorylation of PKCδT505 is negated by inhibiting autophagy; (ii) shear-induced p-eNOSS1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (e.g., bryostatin) or genetic (e.g., CA-PKCδ) activation of PKCδT505 and (iii) pharmacological (e.g., rottlerin) and genetic (e.g., PKCδ siRNA) PKCδ inhibition prevents shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway upon autophagy compromise were revealed in human arterial endothelial cells. Conclusion Targeted reactivation of purinergic signaling and/or PKCδ has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.