ABSTRACT
BACKGROUND AND PURPOSE: Remote ischaemic preconditioning (rIPC) for cardioprotection is severely impaired in diabetes, and therapeutic options to restore it are lacking. The vascular endothelium plays a key role in rIPC. Given that the activity of endothelial nitric oxide synthase (eNOS) is inhibited by proline-rich tyrosine kinase 2 (Pyk2), we hypothesized that pharmacological Pyk2 inhibition could restore eNOS activity and thus restore remote cardioprotection in diabetes. EXPERIMENTAL APPROACH: New Zealand obese (NZO) mice that demonstrated key features of diabetes were studied. The consequence of Pyk2 inhibition on endothelial function, rIPC and infarct size after myocardial infarction were evaluated. The impact of plasma from mice and humans with or without diabetes was assessed in isolated buffer perfused murine hearts and aortic rings. KEY RESULTS: Plasma from nondiabetic mice and humans, both subjected to rIPC, caused remote tissue protection. Similar to diabetic humans, NZO mice demonstrated endothelial dysfunction. NZO mice had reduced circulating nitrite levels, elevated arterial blood pressure and a larger infarct size after ischaemia and reperfusion than BL6 mice. Pyk2 increased the phosphorylation of eNOS at its inhibitory site (Tyr656), limiting its activity in diabetes. The cardioprotective effects of rIPC were abolished in diabetic NZO mice. Pharmacological Pyk2 inhibition restored endothelial function and rescued cardioprotective effects of rIPC. CONCLUSION AND IMPLICATIONS: Endothelial function and remote tissue protection are impaired in diabetes. Pyk2 is a novel target for treating endothelial dysfunction and restoring cardioprotection through rIPC in diabetes.
ABSTRACT
BACKGROUND: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. METHODS: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RESULTS: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. CONCLUSIONS: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.
Subject(s)
Hydrogen Sulfide , Telomerase , Animals , Humans , Mice , Cellular Senescence , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/metabolism , Endothelial Cells/metabolism , Hydrogen Sulfide/metabolism , Telomerase/genetics , Telomerase/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Macrophages are plastic and heterogeneous immune cells that adapt pro- or anti-inflammatory phenotypes upon exposure to different stimuli. Even though there has been evidence supporting a crosstalk between coagulation and innate immunity, the way in which protein components of the hemostasis pathway influence macrophages remains unclear. We investigated the effect of thrombin on macrophage polarization. On the basis of gene expression and cytokine secretion, our results suggest that polarization with thrombin induces an anti-inflammatory, M2-like phenotype. In functional studies, thrombin polarization promoted oxLDL phagocytosis by macrophages, and conditioned medium from the same cells increased endothelial cell proliferation. There were, however, clear differences between the classical M2a polarization and the effects of thrombin on gene expression. Finally, the deletion and inactivation of secreted modular Ca2+-binding protein 1 (SMOC1) attenuated phagocytosis by thrombin-stimulated macrophages, a phenomenon revered by the addition of recombinant SMOC1. Manipulation of SMOC1 levels also had a pronounced impact on the expression of TGF-ß-signaling-related genes. Taken together, our results show that thrombin induces an anti-inflammatory macrophage phenotype with similarities as well as differences to the classical alternatively activated M2 polarization states, highlighting the importance of tissue levels of SMOC1 in modifying thrombin-induced macrophage polarization.
Subject(s)
Macrophages , Thrombin , Animals , Anti-Inflammatory Agents/metabolism , Macrophage Activation , Macrophages/metabolism , Mice , Phagocytosis , Thrombin/pharmacologyABSTRACT
BACKGROUND: POR (cytochrome P450 reductase) provides electrons for the catalytic activity of the CYP (cytochrome P450) monooxygenases. CYPs are dual-function enzymes as they generate protective vasoactive mediators derived from polyunsaturated fatty acids but also reactive oxygen species. It is not known in which conditions the endothelial POR/CYP system is beneficial versus deleterious. Here, the activity of all CYP enzymes was eliminated in the vascular endothelium to examine its impact on vascular function. METHODS: An endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR-/-) was generated. Vascular function was studied by organ chamber experiments. eNOS (endothelial nitric oxide synthase) activity was accessed by heavy arginine/citrulline LC-MS/MS detection and phosphorylation of serine1177 in aortic rings. CYP-derived epoxyeicosatrienoic acids and prostanoids were measured by LC-MS/MS. Gene expression of aorta and endothelial cells was profiled by RNA sequencing. Blood pressure was measured by telemetry. RESULTS: Acetylcholine-induced endothelium-dependent relaxation was attenuated in isolated vessels of ecPOR-/- as compared with control mice. Additionally, ecPOR-/- mice had attenuated eNOS activity and eNOS/AKT phosphorylation. POR deletion reduced endothelial stores of CYP-derived epoxyeicosatrienoic acids but increased vascular prostanoids. This phenomenon was paralleled by the induction of genes implicated in eicosanoid generation. In response to Ang II (angiotensin II) infusion, blood pressure increased significantly more in ecPOR-/- mice. Importantly, the cyclooxygenase inhibitor Naproxen selectively lowered the Ang II-induced hypertension in ecPOR-/- mice. CONCLUSIONS: POR expression in endothelial cells maintains eNOS activity and its loss results in an overactivation of the vasoconstrictor prostanoid system. Through these mechanisms, loss of endothelial POR induces vascular dysfunction and hypertension.
Subject(s)
Hypertension , NADPH-Ferrihemoprotein Reductase , Animals , Chromatography, Liquid , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Hypertension/chemically induced , Hypertension/metabolism , Mice , Mice, Knockout , NADPH-Ferrihemoprotein Reductase/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Prostaglandins/metabolism , Tandem Mass Spectrometry , VasodilationABSTRACT
Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and ß3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-ß signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.
Subject(s)
Blood Platelets/metabolism , Diabetes Mellitus, Type 2/metabolism , Osteonectin/metabolism , Thrombin/metabolism , Adult , Animals , Blood Platelets/cytology , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Platelet Activation , Platelet AggregationABSTRACT
AIMS: Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity. METHODS AND RESULTS: In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium-intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases, i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and down-regulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition. CONCLUSIONS: VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension.
Subject(s)
Aniline Compounds/therapeutic use , Antihypertensive Agents/therapeutic use , Diabetes Mellitus/drug therapy , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Enzyme Inhibitors/therapeutic use , Hypertension/drug therapy , Nitric Oxide Synthase Type III/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 3/antagonists & inhibitors , Sulfonic Acids/therapeutic use , Animals , Blood Pressure/drug effects , Cells, Cultured , Diabetes Mellitus/enzymology , Diabetes Mellitus/genetics , Diabetes Mellitus/physiopathology , Disease Models, Animal , Endothelial Cells/enzymology , Endothelium, Vascular/enzymology , Endothelium, Vascular/physiopathology , Humans , Hypertension/enzymology , Hypertension/genetics , Hypertension/physiopathology , Mice, Inbred C57BL , Mice, Transgenic , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/genetics , Phosphorylation , Receptor-Like Protein Tyrosine Phosphatases, Class 3/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 3/metabolism , Signal Transduction , Treatment Outcome , United StatesABSTRACT
Diabetes is associated with platelet hyper-reactivity and enhanced risk of thrombosis development. Here we compared protein expression in platelets from healthy donors and diabetic patients to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analyses identified cyclin Y (CCNY) in platelets and its reduced expression in platelets from diabetic patients, a phenomenon that could be attributed to the increased activity of calpains. To determine the role of CCNY in platelets, mice globally lacking the protein were studied. CCNY-/- mice demonstrated lower numbers of circulating platelets but platelet responsiveness to thrombin and a thromboxane A2 analogue were comparable with that of wild-type mice, as was agonist-induced α and dense granule secretion. CCNY-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in "outside in" integrin signalling. This phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of ß3 integrin. Taken together we have shown that CCNY is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling associated with thrombin stimulation.
Subject(s)
Blood Platelets/metabolism , Cyclins/genetics , Integrins/metabolism , Adult , Animals , Cyclins/metabolism , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Platelet Activation/genetics , Platelet Adhesiveness/genetics , Platelet Aggregation/genetics , Signal Transduction/genetics , Young AdultABSTRACT
Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein-protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein-protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS-PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.
Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Nitric Oxide Synthase Type III/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Biological Availability , Cell Line , Gene Expression Regulation, Enzymologic/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Nitric Oxide , Nitric Oxide Synthase Type III/genetics , Piperazines/pharmacology , Plasminogen Activator Inhibitor 1/genetics , Protein Binding , Vasodilation/drug effects , Vasodilation/physiology , para-Aminobenzoates/pharmacologyABSTRACT
Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.
Subject(s)
Amino Acid Substitution , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/metabolism , Pyruvate Kinase/metabolism , Animals , Cells, Cultured , Endothelial Cells , Homeostasis , Humans , Male , Mice , Nitric Oxide Synthase Type III/genetics , Oxidation-Reduction , Pentose Phosphate Pathway , Protein BindingABSTRACT
Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress-induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress-induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress-induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.
Subject(s)
Adrenomedullin/metabolism , Blood Pressure , Endothelium, Vascular , Second Messenger Systems , Stress, Mechanical , Animals , Cyclic AMP/metabolism , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Human Umbilical Vein Endothelial Cells , Humans , Hypertension/metabolism , Hypertension/pathology , Hypertension/physiopathology , Ion Channels/metabolism , Male , Mice , Mice, Knockout , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolismABSTRACT
BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.
Subject(s)
Atherosclerosis/enzymology , Carotid Arteries/enzymology , Carotid Artery Diseases/enzymology , Cystathionine gamma-Lyase/metabolism , ELAV-Like Protein 1/metabolism , Endothelial Cells/enzymology , Hydrogen Sulfide/metabolism , Plaque, Atherosclerotic , Aged , Aged, 80 and over , Animals , Atherosclerosis/genetics , Atherosclerosis/pathology , Atherosclerosis/prevention & control , Carotid Arteries/pathology , Carotid Artery Diseases/genetics , Carotid Artery Diseases/pathology , Carotid Artery Diseases/prevention & control , Cathepsins/metabolism , Cell Adhesion , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cystathionine gamma-Lyase/deficiency , Cystathionine gamma-Lyase/genetics , Disease Models, Animal , Disease Progression , ELAV-Like Protein 1/genetics , Endothelial Cells/pathology , Female , HEK293 Cells , Humans , Male , Mice, Inbred C57BL , Mice, Knockout, ApoE , Middle Aged , Monocytes/metabolism , Monocytes/pathology , Phosphorylation , Protein Processing, Post-Translational , Signal TransductionABSTRACT
Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells (mESC) and occurs after release from serum and leukemia inhibitory factor (LIF). Here we show that after release from pluripotency, a subpopulation of mESC, kept in the naive state by 2i/LIF, expresses endothelial nitric oxide synthase (eNOS) and endogenously synthesizes NO. This eNOS/NO-positive subpopulation (ESNO+) expresses mesendodermal markers and is more efficient in the generation of cardiovascular precursors than eNOS/NO-negative cells. Mechanistically, production of endogenous NO triggers rapid Hdac2 S-nitrosylation, which reduces association of Hdac2 with the transcriptional repression factor Zeb1, allowing mesendodermal gene expression. In conclusion, our results suggest that the interaction between Zeb1, Hdac2, and eNOS is required for early mesendodermal differentiation of naive mESC.
Subject(s)
Histone Deacetylase 2/metabolism , Mouse Embryonic Stem Cells/cytology , Myocardium/cytology , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/biosynthesis , Zinc Finger E-box-Binding Homeobox 1/metabolism , Animals , Cell Differentiation/physiology , Cell Line, Tumor , HeLa Cells , Humans , Leukemia Inhibitory Factor/metabolism , Mice , Mouse Embryonic Stem Cells/metabolism , Myocardium/metabolismABSTRACT
Zebrafish is a widely used model to evaluate genetic variants and modifiers that can cause heart muscle diseases. Surprisingly, the ß-adrenergic receptor (ß-AR) pathway in zebrafish is not well characterized, although abnormal ß-AR signaling is a major contributor to human heart failure (HF). Chronic ß-AR activation in the attempt to normalize heart function in the failing heart results in a reduction of the ß-ARs expression and receptor desensitization, largely mediated through G-protein coupled receptor kinase 2 (GRK2) upregulation. This in turn leads to further deterioration of heart function and progression towards HF. This study seeks to systematically characterize the function of the ß-AR signaling in developing and adult zebrafish to ultimately assess the ability to induce HF through chronic ß-AR activation by isoproterenol (ISO) as established in the mouse model. Larval hearts first responded to ISO by 3dpf, in concordance with robust expression of key components of the ß-AR signaling pathway. Although ISO-induced ß1-AR and ß2-AR isoform upregulation persisted, chronic ISO stimulation for 5d caused systolic cardiac dysfunction concurrently with maximal expression of G-protein-coupled receptor kinase-2 (GRK2). More consistent to mammalians, adult zebrafish developed significant heart failure in concert with ß1-AR downregulation, and GRK2 and brain natriuretic peptide (BNP) upregulation in response to prolonged, 14d ISO-stimulation. This was accompanied by significant cell death and inflammation without detectable fibrosis. Our study unveils important characteristics of larvae and adult zebrafish hearts pertaining to ß-AR signaling. A lack of ß-AR responsiveness and atypical ß-AR/GRK2 ratios in larval zebrafish should be considered. Adult zebrafish resembled the mammalian situation on the functional and molecular level more closely, but also revealed differences to dysfunctional mammalian hearts, i.e. lack of fibrosis. Our study establishes the first ISO-inducible HF model in adult zebrafish and present critical characteristics of the zebrafish heart essential to be considered when utilizing the zebrafish as a human disease and future drug discovery model.
Subject(s)
Adrenergic beta-Agonists/administration & dosage , Heart/drug effects , Heart/physiopathology , Isoproterenol/administration & dosage , Adrenergic beta-Agonists/adverse effects , Animals , Calcium/metabolism , Disease Models, Animal , Echocardiography , Heart Diseases/diagnostic imaging , Heart Diseases/etiology , Heart Diseases/pathology , Heart Diseases/physiopathology , Heart Function Tests , Isoproterenol/adverse effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , ZebrafishSubject(s)
Insulin Resistance , Endothelium , Humans , Insulin , Nitric Oxide Synthase Type III , Receptor, Insulin , Signal TransductionABSTRACT
We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α-activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B-deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition.
ABSTRACT
Endothelial nitric oxide synthase (eNOS) plays an essential role in the regulation of endothelial function and acts as a master regulator of vascular tone and homeostasis through the generation of the gasotransmitter nitric oxide (NO). The complex network of events mediating efficient NO synthesis is regulated by post-translational modifications and protein-protein interactions. Dysregulation of these mechanisms induces endothelial dysfunction, a term often used to refer to reduced NO bioavailability and consequent alterations in endothelial function, that are a hallmark of many cardiovascular diseases. Endothelial dysfunction is linked to eNOS uncoupling, which consists of a switch from the generation of NO to the generation of superoxide anions and hydrogen peroxide. This review provides an overview of the eNOS signalosome, integrating past and recently described protein-protein interactions that have been shown to play a role in the modulation of eNOS activity with implications for cardiovascular pathophysiology. The mechanisms underlying eNOS uncoupling and clinically relevant strategies that were adopted to influence them are also discussed.
Subject(s)
Endothelium, Vascular/metabolism , Nitric Oxide Synthase Type III/metabolism , Animals , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/physiology , Humans , Nitric Oxide/metabolism , Protein Interaction Domains and Motifs/physiology , Signal Transduction/physiologyABSTRACT
The contribution of endothelial-derived miR-17â¼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17â¼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17â¼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17â¼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.
Subject(s)
Frizzled Receptors/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , MicroRNAs/metabolism , Multigene Family/physiology , Neovascularization, Physiologic/physiology , Wnt Signaling Pathway/physiology , Animals , Frizzled Receptors/genetics , Ischemia/genetics , Ischemia/metabolism , Ischemia/pathology , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Mice , Mice, Knockout , MicroRNAs/geneticsABSTRACT
Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells.
Subject(s)
Endothelial Cells/metabolism , HSP90 Heat-Shock Proteins/metabolism , Nitric Oxide Synthase Type III/metabolism , Proteins/metabolism , Animals , COS Cells , Cell Line , Cells, Cultured , Chlorocebus aethiops , Chromatography, Liquid , Endothelial Cells/drug effects , Enzyme Activation , HEK293 Cells , Humans , Immunoblotting , Nitric Oxide/metabolism , Phosphorylation , Protein Binding , Proteins/genetics , RNA Interference , Serine/metabolism , Signal Transduction/drug effects , Tandem Mass Spectrometry , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
OBJECTIVE: Telmisartan, an angiotensin II type 1 receptor blocker, and amlodipine, a calcium channel blocker, are antihypertensive agents clinically used as monotherapy or in combination. They exert beneficial cardiovascular effects independently of blood pressure lowering and classic mechanisms of action. In this study, we investigate molecular mechanisms responsible for the off-target effects of telmisartan and telmisartan-amlodipine in endothelial cells (ECs), using an unbiased genomic approach. APPROACH AND RESULTS: In human umbilical vein ECs, microarray analysis of gene expression followed by pathway enrichment analysis and quantitative polymerase chain reaction validation revealed that telmisartan modulates the expression of key genes responsible for cell cycle progression and apoptosis. Amlodipine's effect was similar to control. ECs exposed to telmisartan, but not amlodipine, losartan, or valsartan, exhibited a dose-dependent impairment of cell growth and failed to enter the S-phase of the cell cycle. Similarly, telmisartan inhibited proliferation in COS-7 cells lacking the angiotensin II type 1 receptor. In telmisartan-treated ECs, phosphorylation and activation of Akt, as well as MDM2, were reduced, leading to accumulation of p53 in the nucleus, where it represses the transcription of cell cycle-promoting genes. Phosphorylation of glycogen synthase kinase-3ß was also reduced, resulting in rapid proteolytic turnover of CyclinD1. Telmisartan induced downregulation of proapoptotic genes and protected ECs from serum starvation-induced and 7-ketocholesterol-induced apoptosis. CONCLUSIONS: Telmisartan exerts antiproliferative and antiapoptotic effects in ECs. This may account for the improved endothelial dysfunction observed in the clinical setting.
Subject(s)
Amlodipine/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Benzimidazoles/pharmacology , Benzoates/pharmacology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Hypertension/drug therapy , Apoptosis/drug effects , Blood Proteins/pharmacology , Cell Cycle Proteins/genetics , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Combinations , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Ketocholesterols/pharmacology , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , TelmisartanABSTRACT
RATIONALE: Phosphoinositide 3-kinase (PI3K)gamma is expressed in hematopoietic cells, endothelial cells (ECs), and cardiomyocytes and regulates different cellular functions relevant to inflammation, tissue remodeling and cicatrization. Recently, PI3Kgamma inhibitors have been indicated for the treatment of chronic inflammatory/autoimmune diseases and atherosclerosis. OBJECTIVE: We aimed to determine PI3Kgamma contribution to the angiogenic capacity of ECs and the effect of PI3Kgamma inhibition on healing of myocardial infarction (MI). METHODS AND RESULTS: Human umbilical ECs were treated with a selective PI3Kgamma inhibitor, AS605240, or a pan-phosphoinositide 3-kinases inhibitor, LY294002. Both inhibitory treatments and small interfering RNA-mediated PI3Kgamma knockdown strongly impaired ECs angiogenic capacity, because of suppression of the PI3K/Akt and mitogen-activated protein kinase pathways. Constitutive activation of Akt rescued the angiogenic defect. Reparative angiogenesis was studied in vivo in a model of MI. AS605240 did not affect MI-induced PI3Kgamma upregulation, whereas it suppressed Akt activation and downstream signaling. AS605240 strongly reduced inflammation, enhanced cardiomyocyte apoptosis, and impaired survival and proliferation of ECs in peri-infarct zone, which resulted in defective reparative neovascularization. As a consequence, AS605240-treated MI hearts showed increased infarct size and impaired recovery of left ventricular function. Similarly, PI3Kgamma-deficient mice showed impaired reparative neovascularization, enhanced cardiomyocyte apoptosis and marked deterioration of cardiac function following MI. Mice expressing catalytically inactive PI3Kgamma also failed to mount a proper neovascularization, although cardiac dysfunction was similar to wild-type controls. CONCLUSIONS: PI3Kgamma expression and catalytic activity are involved at different levels in reparative neovascularization and healing of MI.