Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function.

Fetal lung fibroblasts contribute dynamic infrastructure for the developing lung. These cells undergo dynamic mechanical transitions, including cyclic stretch and spreading, which are integral to lung growth in utero. We investigated the role of the nuclear envelope protein emerin in cellular responses to these dynamic mechanical transitions. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, fibroblasts that were acutely downregulated for emerin showed incomplete reorientation of both nuclei and actin cytoskeleton. Emerin-downregulated fibroblasts were also aberrantly circular in contrast to the spindle-shaped controls and exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus. Emerin knockdown was also associated with reduced myosin light chain phosphorylation during cell spreading. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and production. These findings indicate that nuclear-cytoskeletal coupling serves a role in the dynamic regulation of cytoskeletal structure and function and may also impact the transmission of traction force to the extracellular matrix microenvironment.

ZMPSTE24 Regulates SARS-CoV-2 Spike Protein-enhanced Expression of Endothelial PAI-1.

Endothelial dysfunction is implicated in the thrombotic events reported in patients with coronavirus disease (COVID-19), but the underlying molecular mechanisms are unknown. Circulating levels of the coagulation cascade activator PAI-1 are substantially higher in patients with COVID-19 with severe respiratory dysfunction than in patients with bacterial sepsis and acute respiratory distress syndrome. Indeed, the elevation of PAI-1 is recognized as an early marker of endothelial dysfunction. Here, we report that the rSARS-CoV-2-S1 (recombinant severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] viral envelope spike) glycoprotein stimulated robust production of PAI-1 by human pulmonary microvascular endothelial cells (HPMECs). We examined the role of protein degradation in this SARS-CoV-2-S1 induction of PAI-1 and found that the proteasomal degradation inhibitor bortezomib inhibited SARS-CoV-2-S1-mediated changes in PAI-1. Our data further show that bortezomib upregulated KLF2, a shear-stress-regulated transcription factor that suppresses PAI-1 expression. Aging and metabolic disorders are known to increase mortality and morbidity in patients with COVID-19. We therefore examined the role of ZMPSTE24 (zinc metallopeptidase STE24), a metalloprotease with a demonstrated role in host defense against RNA viruses that is decreased in older individuals and in metabolic syndrome, in the induction of PAI-1 in HPMECs by SARS-CoV-2-S1. Indeed, overexpression of ZMPSTE24 blunted enhancement of PAI-1 production in spike protein-exposed HPMECs. In addition, we found that membrane expression of the SARS-CoV-2 entry receptor ACE2 was reduced by ZMPSTE24-mediated cleavage and shedding of the ACE2 ectodomain, leading to accumulation of ACE2 decoy fragments that may bind SARS-CoV-2. These data indicate that decreases in ZMPSTE24 with age and comorbidities may increase vulnerability to vascular endothelial injury by SARS-CoV-2 viruses and that enhanced production of endothelial PAI-1 might play role in prothrombotic events in patients with COVID-19.

Endothelial-Specific Overexpression of Histone Deacetylase 2 Protects Mice against Endothelial Dysfunction and Atherosclerosis.

BACKGROUND/AIMS: We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in protecting endothelial cells from oxidized low-density lipoprotein (OxLDL)-induced injury. In this study, we examined the effects of endothelial-specific HDAC2 overexpression on endothelial-dependent vasorelaxation and atherogenesis in vivo. METHODS: Endothelial-specific HDAC2-overexpressing transgenic mice (HDAC2-Tg) were generated under control of the Tie2 promoter. An atherosclerosis model was produced by injecting HDAC2-Tg and wild-type (WT) mice with adeno-associated virus encoding a PCSK9 gain-of-function mutant under control of a liver-specific promoter and feeding them a high-fat diet for 12 weeks. Aortic stiffness in vivo was determined by measuring pulse wave velocity. Wire myography was used to measure endothelium dependent (acetylcholine) and independent (sodium nitroprusside) relaxation in isolated mice aortas. Atherosclerotic plaque burden in aortas was determined by Oil Red O staining and protein expression was determined by western blotting. RESULTS: At baseline, HDAC2-Tg mice had normal mean arterial blood pressure (MAP) and body weight, but pulse wave velocity (PWV), an inverse measure of vascular health and stiffness, was decreased, suggesting that their vessels were more compliant. Moreover, basal nitric oxide production was enhanced in the vessels of HDAC2-Tg mice as compared to that in WT controls, although no significant differences in acetylcholine (endothelial component)- or sodium nitroprusside (non-endothelial component)-mediated relaxation were observed. However, after exposure to OxLDL, aortas from HDAC2-Tg mice exhibited greater acetylcholine-induced relaxation than did those from WT mice. Thus, endothelial-specific vasodilator production was enhanced despite oxidative injury. Atherosclerosis induction in WT mice led to a significant increase in PWV, but in HDAC2-Tg mice, PWV and MAP remained unchanged. Further, aortic rings from HDAC2-Tg exhibited better endothelial-dependent vascular relaxation than did those from WT mice, but not when treated with nitric oxide synthase inhibitor L-NAME. Finally, plaque burden, determined by Oil red O staining, was significantly increased in WT, but not HDAC2-Tg mice, subjected to the atherogenic model. Deletion of endothelial HDAC2 led to impaired endothelial cell-dependent vascular relaxation and increased PWV, compared with those in littermate controls. CONCLUSION: HDAC2 protects against endothelial dysfunction and atherogenesis induced by oxidized lipids. Hence, overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis. HDAC2-Tg mice provide an opportunity to determine the role of endothelial HDAC2 in vascular endothelial homeostasis.

Histone deacetylase 6 inhibitor tubastatin A attenuates angiotensin II-induced hypertension by preventing cystathionine γ-lyase protein degradation.

Cystathionine γ-lyase (CSEγ) is a hydrogen sulfide (H2S)-producing enzyme. Endothelial H2S production can mediate vasodilatory effects, contributing to the alleviation of hypertension (high blood pressure). Recent studies have suggested a role of histone deacetylase 6 (HDAC6) in hypertension, although its underlying mechanisms are poorly understood. Here, we addressed the potential regulation of CSEγ by HDAC6 in angiotensin II (AngII)-induced hypertension and its molecular details focusing on CSEγ posttranslational modification. Treatment of mice with a selective HDAC6 inhibitor tubastatin A (TubA) alleviated high blood pressure and vasoconstriction induced by AngII. Cotreatment of the aorta and human aortic endothelial cells with TubA recovered AngII-mediated decreased H2S levels. AngII treatment upregulated HDAC6 mRNA and protein expression, but conversely downregulated CSEγ protein. Notably, potent HDAC6 inhibitors and HDAC6 siRNA as well as a proteasomal inhibitor increased CSEγ protein levels and blocked the downregulatory effect of AngII on CSEγ. In contrast, other HDAC isoforms-specific inhibitors and siRNAs did not show such blocking effects. Transfected CSEγ protein levels were also reciprocally regulated by AngII and TubA, and were reduced by wild-type, but not by deacetylase-deficient, HDAC6. Moreover, TubA significantly increased both protein stability and K73 acetylation level of CSEγ. Consistent with these results, AngII induced CSEγ ubiquitination and degradation, which was inhibited by TubA. Our results indicate that AngII promoted HDAC6-dependent deacetylation of CSEγ at K73 residue, leading to its ubiquitin-mediated proteolysis, which underlies AngII-induced hypertension. Overall, this study suggests that upregulation of CSEγ and H2S through HDAC6 inhibition may be considered as a valid strategy for preventing the progression of hypertension.

Hypoxia Triggers SENP1 (Sentrin-Specific Protease 1) Modulation of KLF15 (Kruppel-Like Factor 15) and Transcriptional Regulation of Arg2 (Arginase 2) in Pulmonary Endothelium.

OBJECTIVE: KLF15 (Kruppel-like factor 15) has recently been shown to suppress activation of proinflammatory processes that contribute to atherogenesis in vascular smooth muscle, however, the role of KLF15 in vascular endothelial function is unknown. Arginase mediates inflammatory vasculopathy and vascular injury in pulmonary hypertension. Here, we tested the hypothesis that KLF15 is a critical regulator of hypoxia-induced Arg2 (arginase 2) transcription in human pulmonary microvascular endothelial cells (HPMEC). APPROACH AND RESULTS: Quiescent HPMEC express ample amounts of full-length KLF15. HPMECs exposed to 24 hours of hypoxia exhibited a marked decrease in KLF15 protein levels and a reciprocal increase in Arg2 protein and mRNA. Chromatin immunoprecipitation indicated direct binding of KLF15 to the Arg2 promoter, which was relieved with HPMEC exposure to hypoxia. Furthermore, overexpression of KLF15 in HPMEC reversed hypoxia-induced augmentation of Arg2 abundance and arginase activity and rescued nitric oxide (NO) production. Ectopic KLF15 also reversed hypoxia-induced endothelium-mediated vasodilatation in isolated rat pulmonary artery rings. Mechanisms by which hypoxia regulates KLF15 abundance, stability, and compartmentalization to the nucleus in HPMEC were then investigated. Hypoxia triggered deSUMOylation of KLF15 by SENP1 (sentrin-specific protease 1), and translocation of KLF15 from nucleus to cytoplasm. CONCLUSIONS: KLF15 is a critical regulator of pulmonary endothelial homeostasis via repression of endothelial Arg2 expression. KLF15 abundance and nuclear compartmentalization are regulated by SUMOylation/deSUMOylation-a hypoxia-sensitive process that is controlled by SENP1. Strategies including overexpression of KLF15 or inhibition of SENP1 may represent novel therapeutic targets for pulmonary hypertension.

Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition.

We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.

Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6.

Endothelial cystathionine γ-lyase (CSEγ) contributes to cardiovascular homeostasis, mainly through production of H2S. However, the molecular mechanisms that control CSEγ gene expression in the endothelium during cardiovascular diseases are unclear. The aim of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial CSEγ. Reduced CSEγ mRNA expression and protein abundance were observed in human aortic endothelial cells (HAEC) exposed to oxidized LDL (OxLDL) and in aortas from atherogenic apolipoprotein E knockout (ApoE-/-) mice fed a high-fat diet compared with controls. Intact murine aortic rings exposed to OxLDL (50 µg/ml) for 24 h exhibited impaired endothelium-dependent vasorelaxation that was blocked by CSEγ overexpression or the H2S donor NaHS. CSEγ expression was upregulated by pan-HDAC inhibitors and by class II-specific HDAC inhibitors, but not by other class-specific inhibitors. The HDAC6 selective inhibitor tubacin and HDAC6-specific siRNA increased CSEγ expression and blocked OxLDL-mediated reductions in endothelial CSEγ expression and CSEγ promoter activity, indicating that HDAC6 is a specific regulator of CSEγ expression. Consistent with this finding, HDAC6 mRNA, protein expression, and activity were upregulated in OxLDL-exposed HAEC, but not in human aortic smooth muscle cells. HDAC6 protein levels in aortas from high-fat diet-fed ApoE-/- mice were comparable to those in controls, whereas HDAC6 activity was robustly upregulated. Together, our findings indicate that HDAC6 is upregulated by atherogenic stimuli via posttranslational modifications and is a critical regulator of CSEγ expression in vascular endothelium. Inhibition of HDAC6 activity may improve endothelial function and prevent or reverse the development of atherosclerosis.NEW & NOTEWORTHY Oxidative injury to endothelial cells by oxidized LDL reduced cystathionine γ-lyase (CSEγ) expression and H2S production, leading to endothelial dysfunction, which was prevented by histone deacetylase 6 (HDAC6) inhibition. Our data suggest HDAC6 as a novel therapeutic target to prevent the development of atherosclerosis.

Melanopsin mediates light-dependent relaxation in blood vessels.

Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. ß-Adrenergic receptor kinase 1 (ßARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.

MPST but not CSE is the primary regulator of hydrogen sulfide production and function in the coronary artery.

Hydrogen sulfide (H2S) has emerged as an important gasotransmitter in the vasculature. In this study, we tested the hypothesis that H2S contributes to coronary vasoregulation and evaluated the physiological relevance of two sources of H2S, namely, cystathionine-γ-lyase (CSE) and 3-mercaptypyruvate sulfertransferase (MPST). MPST was detected in human coronary artery endothelial cells as well as rat and mouse coronary artery; CSE was not detected in the coronary vasculature. Rat coronary artery homogenates produced H2S through the MPST pathway but not the CSE pathway in vitro. In vivo coronary vasorelaxation response was similar in CSE knockout mice, wild-type mice (WT), and WT mice treated with the CSE inhibitor propargylglycine, suggesting that CSE-produced H2S does not have a significant role in coronary vasoregulation in vivo. Ex vivo, the MPST substrate 3-mercaptopyruvate (3-MP) and H2S donor sodium hydrosulfide (NaHS) elicited similar coronary vasoreactivity responses. Pyruvate did not have any effects on vasoreactivity. The vasoactive effect of H2S appeared to be nitric oxide (NO) dependent: H2S induced coronary vasoconstriction in the presence of NO and vasorelaxation in its absence. Maximal endothelial-dependent relaxation was intact after 3-MP and NaHS induced an increase in preconstriction tone, suggesting that endothelial NO synthase activity was not significantly inhibited. In vitro, H2S reacted with NO, which may, in part explain the vasoconstrictive effects of 3-MP and NaHS. Taken together, these data show that MPST rather than CSE generates H2S in coronary artery, mediating its effects through direct modulation of NO. This has important implications for H2S-based therapy in healthy and diseased coronary arteries.

OxLDL triggers retrograde translocation of arginase2 in aortic endothelial cells via ROCK and mitochondrial processing peptidase.

RATIONALE: Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking. OBJECTIVE: To test the hypotheses that OxLDL triggers reverse translocation of mitochondrial arginase 2 (Arg2) to cytosol and Arg2 activation, and that this process is dependent on mitochondrial processing peptidase, lectin-like OxLDL receptor-1 receptor, and rho kinase. METHODS AND RESULTS: OxLDL-triggered translocation of Arg2 from mitochondria to cytosol in human aortic endothelial cells and in murine aortic intima with a concomitant rise in arginase activity. All of these changes were abolished by inhibition of mitochondrial processing peptidase or by its siRNA-mediated knockdown. Rho kinase inhibition and the absence of the lectin-like OxLDL receptor-1 in knockout mice also ablated translocation. Aminoterminal sequencing of Arg2 revealed 2 candidate mitochondrial targeting sequences, and deletion of either of these confined Arg2 to the cytoplasm. Inhibitors of mitochondrial processing peptidase or lectin-like OxLDL receptor-1 knockout attenuated OxLDL-mediated decrements in endothelial-specific NO production and increases in superoxide generation. Finally, Arg2(-/-) mice bred on an ApoE(-/-) background showed reduced plaque load, reduced reactive oxygen species production, enhanced NO, and improved endothelial function when compared with ApoE(-/-) controls. CONCLUSIONS: These data demonstrate dual distribution of Arg2, a protein with an unambiguous mitochondrial targeting sequence, in mammalian cells, and its reverse translocation to cytoplasm by alterations in the extracellular milieu. This novel molecular mechanism drives OxLDL-mediated arginase activation, endothelial NOS uncoupling, endothelial dysfunction, and atherogenesis.

NEDDylation promotes endothelial dysfunction: a role for HDAC2.

Emerging evidence strongly supports a role for HDAC2 in the transcriptional regulation of endothelial genes and vascular function. We have recently demonstrated that HDAC2 reciprocally regulates the transcription of Arginase2, which is itself a critical modulator of endothelial function via eNOS. Moreover HDAC2 levels are decreased in response to the atherogenic stimulus OxLDL via a mechanism that is apparently dependent upon proteasomal degradation. NEDDylation is a post-translational protein modification that is tightly linked to ubiquitination and thereby protein degradation. We propose that changes in NEDDylation may modulate vascular endothelial function in part through alterations in the proteasomal degradation of HDAC2. In HAEC, OxLDL exposure augmented global protein NEDDylation. Pre-incubation of mouse aortic rings with the NEDDylation activating enzyme inhibitor, MLN4924, prevented OxLDL-induced endothelial dysfunction. In HAEC, MLN enhanced HDAC2 abundance, decreased expression and activity of Arginase2, and blocked OxLDL-mediated reduction of HDAC2. Additionally, HDAC2 was shown to be a substrate for NEDD8 conjugation and this interaction was potentiated by OxLDL. Further, HDAC2 levels were reciprocally regulated by ectopic expression of NEDD8 and the de-NEDDylating enzyme SENP8. Our findings indicate that the observed improvement in endothelial dysfunction with inhibition of NEDDylation activating enzyme is likely due to an HDAC2-dependent decrease in Arginase2. NEDDylation activating enzyme may therefore be a novel target in endothelial dysfunction and atherogenesis.

Transcriptional regulation of endothelial arginase 2 by histone deacetylase 2.

OBJECTIVE: Arginase 2 (Arg2) is a critical target in atherosclerosis because it controls endothelial nitric oxide, proliferation, fibrosis, and inflammation. Regulators of Arg2 transcription in the endothelium have not been characterized. The goal of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial Arg2 transcription and endothelial function. APPROACH AND RESULTS: The HDAC inhibitor trichostatin A increased levels of Arg2 mRNA, protein, and activity in both human aortic endothelial cells and mouse aortic rings. These changes occurred in both time- and dose-dependent patterns and resulted in Arg2-dependent endothelial dysfunction. Trichostatin A and the atherogenic stimulus oxidized low-density lipoprotein enhanced the activity of common promoter regions of Arg2. HDAC inhibition with trichostatin A also decreased endothelial nitric oxide, and these effects were blunted by arginase inhibition. Nonselective class I HDAC inhibitors enhanced Arg2 expression, whereas the only selective inhibitor that increased Arg2 expression was mocetinostat, a selective inhibitor of HDACs 1 and 2. Additionally, mouse aortic rings preincubated with mocetinostat exhibited dysfunctional relaxation. Overexpression of HDAC2 (but not HDAC 1, 3, or 8) cDNA in human aortic endothelial cells suppressed Arg2 expression in a concentration-dependent manner, and siRNA knockdown of HDAC2 enhanced Arg2 expression. Chromatin immunoprecipitation indicated direct binding of HDAC2 to the Arg2 promoter, and HDAC2 overexpression in human aortic endothelial cells blocked oxidized low-density lipoprotein-mediated activation of the Arg2 promoter. Finally, overexpression of HDAC2 blocked oxidized low-density lipoprotein-mediated vascular dysfunction. CONCLUSIONS: HDAC2 is a critical regulator of Arg2 expression and thereby endothelial nitric oxide and endothelial function. Overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis.

Insights into the arginine paradox: evidence against the importance of subcellular location of arginase and eNOS.

Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.

Increased epidermal growth factor-like ligands are associated with elevated vascular nicotinamide adenine dinucleotide phosphate oxidase in a primate model of atherosclerosis.

OBJECTIVE: To characterize the relationship between the expression of epidermal growth factor (EGF)-like ligands and vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and activity in a primate model of atherosclerosis. METHODS AND RESULTS: Adult male Cynomolgus monkeys were fed a normal or atherogenic (AS) diet for 45 months, after which animals from the AS group were placed on a normal diet for 8 months (regression). The expression of membrane-associated EGF-like ligands was increased in arteries from animals on the AS diet and normalized in the regression group. EGF-like ligands were distributed throughout atherosclerotic vessels but predominantly colocalized with macrophages. Consistent with ligand shedding, circulating heparin-bound EGF was elevated in the plasma of AS monkeys but not in those on regression diet. Atherosclerosis was associated with the activation of EGF receptor signaling. Expression of NADPH oxidase subunits Nox1 and Nox2 but not Nox4 or Nox5 was increased in arteries from monkeys on the AS diet and returned to normal with regression. Levels of Nox1 and Nox2 positively correlated with EGF-like ligands. In cultured monkey smooth muscle cells, treatment with EGF-like ligands increased Nox1 expression and activity. CONCLUSIONS: These data identify EGF-like ligands as potential modulators of atherogenesis, resulting in part from increased vascular NADPH oxidase activity.

Expression and functional significance of NADPH oxidase 5 (Nox5) and its splice variants in human blood vessels.

The expression and functional significance of NADPH oxidase 5 (Nox5) and its five isoforms in vascular cells is poorly understood. The goal of this study was to determine whether Nox5-α, -ß, -δ, -γ, and -ε (short) are expressed in human blood vessels and evaluate their respective functions. Nox5 mRNA and protein were detected in human blood vessels, cultured human vascular smooth muscle (HVSMC) and endothelium, but not fibroblasts. The most abundant isoforms were α and ß, whereas δ and γ were not detected. Nox5-α and -ß produced reactive oxygen species (ROS), but -δ, -γ, and -ε were not catalytically active. Coexpression of the active Nox5 isoforms with inactive Nox5 variants suppressed ROS production, and coimmunoprecipitation revealed that Nox5-ß binds the inactive ε variant, which may account for reduced ROS production. In HVSMC, angiotensin II, endothelin-1 and TNF-α increased endogenous Nox5 mRNA levels, while adenovirus-mediated overexpression of Nox5 promoted p38 MAPK, JAK2, JNK, and ERK1/2 phosphorylation in endothelial cells (EC), but only increased ERK1/2 phosphorylation in HVSMC. At higher levels of Nox5, there was evidence of increased apoptosis in EC, but not in HVSMC, as detected by the presence of cleaved caspase-3 and cleaved poly(ADP-ribose)polymerase. Although catalytically inactive, Nox5-ε potently activated ERK in HVSMC, and increased expression of Nox5-ε promoted HVSMC proliferation. Nox5 is expressed in human blood vessels. The Nox5-α and -ß splice variants are the major isoforms that are expressed and the only variants capable of ROS production. Nox5-ε can inhibit Nox5 activity and activate ERK and HVSMC proliferation.

SUMO1 negatively regulates reactive oxygen species production from NADPH oxidases.

OBJECTIVE: Increased protein SUMOylation (small ubiquitin-related modifier [SUMO]) provides protection from cellular stress, including oxidative stress, but the mechanisms involved are incompletely understood. The NADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS) and oxidative stress, and thus our goal was to determine whether SUMO regulates NADPH oxidase activity. METHODS AND RESULTS: Increased expression of SUMO1 potently inhibited the activity of Nox1 to Nox5. In contrast, inhibition of endogenous SUMOylation with small interfering RNA to SUMO1 or ubiquitin conjugating enzyme 9 or with the inhibitor anacardic acid increased ROS production from human embryonic kidney-Nox5 cells, human vascular smooth muscle cells, and neutrophils. The suppression of ROS production was unique to SUMO1, and it required a C-terminal diglycine and the SUMO-specific conjugating enzyme ubiquitin conjugating enzyme 9. SUMO1 did not modify intracellular calcium or Nox5 phosphorylation but reduced ROS output in an isolated enzyme assay, suggesting direct effects of SUMOylation on enzyme activity. However, we could not detect the presence of SUMO1 conjugation on Nox5 using a variety of approaches. Moreover, the mutation of more than 17 predicted and conserved lysine residues on Nox5 did not alter the inhibitory actions of SUMO1. CONCLUSIONS: Together, these results suggest that SUMO is an important regulatory mechanism that indirectly represses the production of ROS to ameliorate cellular stress.

Calcium/calmodulin-dependent kinase II mediates the phosphorylation and activation of NADPH oxidase 5.

Excessive synthesis of reactive oxygen species contributes to the pathology of many human diseases and originates from changes in the expression and posttranslational regulation of the transmembrane NADPH oxidases (Noxes). Nox5 is a novel Nox isoform whose activity is regulated by intracellular calcium levels. We have reported that the activity and calcium-sensitivity of Nox5 can also be modulated by direct phosphorylation. However, the kinases that phosphorylate Nox5 have not been identified, and thus, the goal of this study was to determine whether calcium-activated kinases such as calcium/calmodulin-dependent kinase II (CAMKII) are involved. We found that Nox5 activity in bovine aortic endothelial cells was suppressed by two doses of the CAMKII inhibitor 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93). In cotransfected COS-7 cells, wild-type and constitutively active CAMKII, but not a dominant-negative, robustly increased basal Nox5 activity. The ability of CAMKII to increase Nox5 activity was also observed with fixed calcium concentrations in an isolated enzyme activity assay. CAMKII did not elevate intracellular calcium or activate other Nox enzymes. In vitro phosphorylation assays revealed that CAMKII can directly phosphorylate Nox5 on Thr494 and Ser498 as detected by phosphorylation state-specific antibodies. Mass spectrometry (MS) analysis revealed the phosphorylation of additional, novel sites at Ser475, Ser502, and Ser675. Of these phosphorylation sites, mutation of only Ser475 to alanine prevented CAMKII-induced increases in Nox5 activity. The ability of CAMKIIα to phosphorylate Ser475 in intact cells was supported by the binding of Nox5 to phosphoprotein-affinity columns and via MS/MS analysis. Together, these results suggest that CAMKII can positively regulate Nox5 activity via the phosphorylation of Ser475.

Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo: A Role for HDAC6 Neddylation.

We previously reported that histone deacetylase 6 (HDAC6) has an important role in endothelial cell (EC) function in vitro. However, whether HDAC6 plays a role in atherogenesis in vivo and the mechanism(s) that control HDAC6 activity/expression in response to atherogenic stimuli are unclear. The goals of this study were to determine whether HDAC6 inhibitor tubacin attenuates atherogenesis and to elucidate specific molecular mechanism(s) that regulate endothelial HDAC6 expression/activity. We evaluated whether administration of tubacin attenuated or reversed the endothelial dysfunction and atherosclerosis induced in mice by a single intraperitoneal injection of adeno-associated viruses encoding liver-target PCSK9 gain-of-function mutant followed by a high fat diet (HFD) for 18 weeks. Tubacin significantly blunted PCSK9-induced increases in pulse wave velocity (index of vascular stiffness and overall vascular health) that are also seen in atherogenic mice. Furthermore, tubacin protected vessels from defective vasorelaxation, as evaluated by acetylcholine-mediated relaxation using wire myograph. Plaque burden defined by Oil Red O staining was also found to be significantly less in mice that received tubacin than in those that received PCSK9 alone. Inhibition of the NEDDylation pathway with MLN4924, an inhibitor of NEDD8-activating enzyme 1 (NAE1), significantly increased HDAC6 activity in HAECs. Interestingly, HDAC6 expression remained unchanged. Further, HAECs exposed to the atherogenic stimulus oxidized low-density lipoprotein (OxLDL) exhibited enhanced HDAC6 activity, which was attenuated by pretreatment with MLN4924. The HDAC6 NEDDylation molecular pathway might regulate genes related to endothelial control of vasomotor tone, reactivity, and atherosclerosis. Tubacin may represent a novel pharmacologic intervention for atherogenesis and other vasculopathies.

Paradoxical activation of endothelial nitric oxide synthase by NADPH oxidase.

OBJECTIVE: Increased formation of reactive oxygen species (ROS) has been identified as a causative factor in endothelial dysfunction by reducing NO bioavailability and uncoupling endothelial nitric oxide synthase (eNOS). However, the specific contribution of ROS to endothelial function is not well understood. METHODS AND RESULTS: A major source of intracellular ROS is the NADPH oxidase (Nox) family of enzymes. The goal of the current study was to directly assess the contribution of NADPH oxidase derived superoxide to eNOS function by expressing Nox5, a single gene product that constitutively produces superoxide within cells. Paradoxically, we found that instead of inhibiting eNOS, coexpression of Nox5 increased NO release from both bovine and human endothelial cells. To establish the functional significance of this observation in intact blood vessels, the endothelium of mouse aorta was transduced with Nox5 or control adenoviruses. Nox5 potently inhibited Ach-induced relaxation and potentiated contractile responses to phenylephrine. In precontracted aortae, acute exposure to superoxide dismutase induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. CONCLUSIONS: These findings suggest that ROS inhibit eNOS function via consumption of NO rather than direct inhibition of enzymatic activity.

Inhibition of TLR4 attenuates vascular dysfunction and oxidative stress in diabetic rats.

UNLABELLED: Hyperglycemia-induced reactive oxygen species (ROS) production plays a major role in the pathogenesis of diabetic vascular dysfunction. However, the underlying mechanisms remain unclear. Toll-like receptor 4 (TLR4), a key component of innate immunity, is known to be activated during diabetes. Therefore, we hypothesize that hyperglycemia activates TLR4 signaling in vascular smooth muscle cells (VSMCs) that triggers ROS production and causes vascular dysfunction. Rat mesenteric VSMCs exposed to high glucose (25 mmol/l) increased TLR4 expression and activated TLR4 signaling via upregulation of myeloid differentiation factor 88 (MyD88). TLR4 inhibitor CLI-095 significantly attenuated elevated levels of ROS and nuclear factor-kappa B (NF-κB) activity in VSMCs exposed to high glucose. Mesenteric arteries from streptozotocin-induced diabetic rats treated with CLI-095 (2 mg/kg/day) intraperitoneally for 2 weeks exhibited reduced ROS generation and attenuated noradrenaline-induced contraction. These results suggest that hyperglycemia-induced ROS generation and NF-κB activation in VSMCs are at least, in part, mediated by TLR4 signaling. Therefore, strategies to block TLR4 signaling pathways pose a promising avenue to alleviate diabetic-induced vascular complications. KEY MESSAGES: High glucose-induced TLR4 activation in vascular smooth muscle cells. Inhibition of TLR4 attenuated high glucose-induced ROS production and NF-κB activity in VSMC. Suppression of TLR4 signaling attenuated mesenteric contraction in diabetic rat.