Juliano L. Sartoretto

Subcellular Targeting and Differential S-Nitrosylation of Endothelial Nitric-oxide Synthase *

Endothelial nitric-oxide synthase (eNOS) undergoes a complex pattern of post-translational modifications that regulate its activity. We have recently reported that eNOS is constitutively S-nitrosylated in endothelial cells and that agonists promote eNOS denitrosylation concomitant with enzyme activation (Erwin, P. A., Lin, A. J., Golan, D. E., and Michel, T. (2005), J. Biol. Chem. 280, 19888–19894). In the present studies, we use mass spectrometry to confirm that the zinc-tetrathiolate cysteines of eNOS are S-nitrosylated. eNOS targeting to the plasma membrane is necessary for enzyme S-nitrosylation, and we report that translocation between cellular compartments is necessary for dynamic eNOS S-nitrosylation. We transfected cells with cDNA encoding wild-type eNOS, which is membrane-targeted, or with acylation-deficient mutant eNOS (Myr–), which is expressed solely in the cytosol. While wild-type eNOS is robustly S-nitrosylated, we found that S-nitrosylation of the Myr– eNOS mutant is nearly abolished. When we transfected cells with a fusion protein in which Myr– eNOS is ligated to the CD8-transmembrane domain (CD8-Myr–), we found that CD8-Myr– eNOS, which does not undergo dynamic subcellular translocation, is hypernitrosylated relative to wild-type eNOS. Furthermore, we found that when endothelial cells transfected with wild-type or CD8-Myr– eNOS are stimulated with eNOS agonist, only wild-type eNOS is denitrosylated; CD8-Myr– eNOS S-nitrosylation is unchanged. These findings indicate that subcellular targeting is a critical determinant of eNOS S-nitrosylation. Finally, we show that eNOS S-nitrosylation can be detected in intact arterial preparations from mouse and that eNOS S-nitrosylation is a dynamic agonist-modulated process in intact blood vessels. These studies suggest that receptor-regulated eNOS S-nitrosylation may represent an important determinant of NO-dependent signaling in the vascular wall.

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis

Nitric oxide (NO) and hydrogen peroxide (H2O2) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu2(FL2E)] to study adult cardiac myocytes from wild-type, eNOSnull, and nNOSnull mice, we discovered that physiological concentrations of H2O2 activate eNOS but not nNOS. H2O2-stimulated eNOS activation depends on phosphorylation of both the AMP-activated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H2O2 biosensor HyPer2 show marked H2O2 synthesis when stimulated by angiotensin II, but not following β-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H2O2, whereas β-adrenergic contractile responses occur independently of H2O2 signaling. These studies establish differential roles for H2O2 in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.

Regulation of Rac1 by Simvastatin in Endothelial Cells: DIFFERENTIAL ROLES OF AMP-ACTIVATED PROTEIN KINASE AND CALMODULIN-DEPENDENT KINASE KINASE-β*

These studies explore the connections between simvastatin, Rac1, and AMP-activated protein kinase (AMPK) pathways in cultured vascular endothelial cells and in arterial preparations isolated from statin-treated mice. In addition to their prominent effects on lipoprotein metabolism, statins can regulate the small GTPase Rac1, and may also affect the phosphorylation of the ubiquitous AMPK. We explored pathways of statin-modulated Rac1 and AMPK activation both in arterial preparations from statin-treated mice as well as in cultured endothelial cells. We treated adult mice with simvastatin daily for 2 weeks and then harvested and analyzed arterial preparations. Simvastatin treatment of mice led to a significant increase in AMPK and LKB1 phosphorylation and to a decrease in protein kinase A activity relative to control animals, associated with a marked increase in Rac1 activation. Exposure of bovine aortic endothelial cells to simvastatin for 24 h strikingly increased GTP-bound Rac1 and led to increased phosphorylation of AMPK as well as the AMPK kinase LKB1. These responses to simvastatin were blocked by mevalonate or geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. Small interfering RNA (siRNA)-mediated knockdown of AMPK abrogated simvastatin-induced Rac1 activation and LKB1 phosphorylation. Importantly, siRNA-mediated knockdown of the key AMPK kinase, calcium/calmodulin-dependent protein kinase kinase β, completely blocked simvastatin-induced endothelial cell migration and also abrogated statin-promoted phosphorylation of AMPK and LKB1, as did pharmacological inhibition with the specific calcium/calmodulin-dependent protein kinase β inhibitor STO-609. Moreover, siRNA-mediated knockdown of Rac1 completely blocked simvastatin-induced LKB1 phosphorylation, but without affecting simvastatin-induced AMPK phosphorylation. These findings establish a key role for simvastatin in activation of a novel Rac1-dependent signaling pathway in the vascular wall.

Caveolin-1 Is a Critical Determinant of Autophagy, Metabolic Switching, and Oxidative Stress in Vascular Endothelium

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1null mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1null mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.

Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMP-activated protein kinase in endothelial cells

Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H2O2 is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H2O2 treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC50 for H2O2-promoted phosphorylation of AMPK is 65 ± 15 μM, within the physiological range of cellular H2O2 concentrations. The Ca2+/calmodulin-dependent protein kinase kinase-β (CaMKKβ) inhibitor STO-609 abolishes H2O2-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKβ abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H2O2 biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H2O2 generation, which is blocked by PEG-catalase. eNOS−/− mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS−/− mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKβ is critically involved in mediating the phosphorylation of AMPK promoted by H2O2 in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H2O2 generation in endothelial cells.

Regulation of VASP phosphorylation in cardiac myocytes: differential regulation by cyclic nucleotides and modulation of protein expression in diabetic and hypertrophic heart

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASPs molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in VASP(null) mice. We found that the beta-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that VASP(null) mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to VASP(+/+) mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart.

Angiotensin-II and MARCKS A HYDROGEN PEROXIDE- AND RAC1-DEPENDENT SIGNALING PATHWAY IN VASCULAR ENDOTHELIUM

Background: The role of the actin-binding protein MARCKS in angiotensin-II signaling is unknown. Results: Biochemical and cell imaging approaches establish that angiotensin-II promotes Rac1- and H2O2-dependent MARCKS phosphorylation and cytoskeleton rearrangement. Conclusion: MARCKS plays a critical role in angiotensin-II signaling. Significance: Angiotensin-II is implicated in vascular physiology and pathophysiology; these studies identify MARCKS as a key determinant of angiotensin-II-modulated responses in the vascular endothelium. MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320–2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H2O2 in the angiotensin-II response. Studies using the H2O2 biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H2O2. We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP2) biosensor revealed that angiotensin-II PIP2 regulation depends on MARCKS and H2O2. siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H2O2 in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.

Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes.

Nitric oxide (NO) and hydrogen peroxide (H2O2) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H2O2–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H2O2 treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca2+ ([Ca2+]i), and document that activity of the L-type Ca2+ channel is necessary for the H2O2-promoted increase in sarcomere shortening and of [Ca2+]i. Using the chemical NO sensor Cu2(FL2E), we discovered that the H2O2-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca2+ channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H2O2-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca2+]i as well as the activation of protein kinase C (PKC). We also found that H2O2-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca2+ channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H2O2-promoted eNOS phosphorylation. Here we present evidence documenting that H2O2-promoted Akt phosphorylation is dependent on activation of the L-type Ca2+ channel, but is independent of PKC. These studies establish key roles for Ca2+- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H2O2.

Novel role for retinol-binding protein 4 in the regulation of blood pressure

Elevated levels of serum retinol‐binding protein 4 (RBP4) contribute to insulin resistance and correlate with increased prevalence of hypertension and myocardial infarction. We sought to determine whether lowering RBP4 would improve blood pressure (BP) and protect against obesity‐ or angiotensin (Ang)‐II‐induced hypertension. Systolic and diastolic BP were lower in the RBP4‐knockout (RBP4‐KO) mice and higher in the RBP4‐overexpressing (RBP4‐Tg) mice compared with BP in the wild‐type (WT) littermates. Carbachol‐induced vasodilatation was increased in arteries from the RBP4‐KO compared with the WT mice and was impaired in the RBP4‐Tg mice. Aortic eNOSSer1177 phosphorylation was enhanced ~50% in the RBP4‐KO mice, with no change in total eNOS protein. Feeding a high‐fat diet increased BP in the RBP4‐KO mice only to the level in the WT mice fed chow and had no effect on aortic eNOSSer1177 phosphorylation. Ang‐II infusion resulted in 22 mmHg lower systolic BP in the RBP4‐KO than in the WT mice, although the relative BP increase over saline infusion was ~30% in both. Ang‐II treatment decreased aortic eNOSSer1177 phosphorylation in the WT and RBP4‐KO mice, but phosphorylation remained higher in the RBP4‐KO mice. Cardiac hypertrophy with Ang‐II treatment was diminished by 56% in the RBP4‐KO mice. Thus, elevated serum RBP4 raises BP and lack of RBP4 reduces it, with commensurate changes in aortic eNOSSer1177 phosphorylation. Lowering RBP4 may reduce BP through enhanced eNOS‐mediated vasodilatation and may be a novel therapeutic approach for hypertension.—Kraus, B. J., Sartoretto, J. L., Polak, P., Hosooka, T., Shiroto, T., Eskurza, I., Lee, S.‐A., Jiang, H., Michel, T., Kahn, B. B. Novel role for retinol‐binding protein 4 in the regulation of blood pressure. FASEB J. 29, 3133‐3140 (2015). www.fasebj.org

Central role for hydrogen peroxide in P2Y1 ADP receptor-mediated cellular responses in vascular endothelium

Significance ADP is best known as a constituent of nucleic acids and for its role in energy metabolism. ADP is also an important signaling molecule that activates cell surface receptors in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease. We studied the signaling pathways activated by ADP receptors in endothelial cells, which form the lining of blood vessels. Vascular disease states cause abnormalities in endothelial function. We identified a critical role for the reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses to ADP. We discovered that P2Y1 ADP receptors promote transactivation of the receptor tyrosine kinase Flt3, suggesting a new mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors cause vascular dysfunction. ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins—including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein—was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl–interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.