LOXL2 inhibition ameliorates pulmonary artery remodeling in pulmonary hypertension.

BACKGROUND: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. METHODS AND RESULTS: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. CONCLUSION: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 could be a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH.

Pro-Con Debate: Should Code Sharing Be Mandatory for Publication?

In this Pro-Con commentary article, we discuss whether or not code sharing should be mandatory for scientific publications. Scientific programming is an increasingly prevalent tool in research. However, there are not unified guidelines for code availability requirements. Some journals require code sharing. Others require code descriptions. Yet others have no policies around code sharing. The Pro side presented here argues that code sharing should be mandatory for all scientific publications involving code. This Pro argument comes in 2 parts. First, any defensible reason for not sharing code is an equally valid a reason for the manuscript itself not being published. Second, lack of code sharing requirements creates 2 tiers of science: one where reproducibility is required and one where it is not. Additionally, the Pro authors suggest that a debate over code sharing is itself a decade out-of-date due to the emerging availability of containerization and virtual environment sharing software. The Pro argument concludes with an appeal that authors release code to make their work more understandable by other researchers. The Con side presented here argues that computer source codes of medical technology equipment should not be subject to mandatory public disclosure. The source code is a crucial part of what makes a particular device unique and allows that device to outperform its competition. The Con authors believe that public disclosure of this proprietary information would destroy all incentives for businesses to develop new and improved technologies. Competition in the free marketplace is what drives companies to constantly improve their products, to develop new and better medical devices. The open disclosure of these "trade secret" details would effectively end that competitive drive. Why invest time, money, and energy developing a "better mousetrap" if your competitors can copy it and produce it the next day?

Extracellular Vesicles From Women With Severe Preeclampsia Impair Vascular Endothelial Function.

BACKGROUND: Preeclampsia (PE) manifesting as hypertension and organ injury is mediated by vascular dysfunction. In biological fluids, extracellular vesicles (EVs) containing microRNA (miRNA), protein, and other cargo released from the placenta may serve as carriers to propagate injury, altering the functional phenotype of endothelial cells. PE has been consistently correlated with increased levels of placenta-derived EVs (pEVs) in maternal circulation. However, whether pEVs impaired endothelial cell function remains to be determined. In this study, we hypothesize that pEVs from pregnant women with severe PE (sPE) impair endothelial function through altered cell signaling. METHODS: We obtained plasma samples from women with sPE (n = 14) and normotensive pregnant women (n = 15) for the isolation of EVs. The total number of EV and pEV contribution was determined by quantifying immunoreactive EV-cluster of designation 63 (CD63) and placental alkaline phosphatase (PLAP) as placenta-specific markers, respectively. Vascular endothelial functional assays were determined by cell migration, electric cell-substrate impedance sensing in human aortic endothelial cells (HAECs), and wire myography in isolated blood vessels, preincubated with EVs from normotensive and sPE women. RESULTS: Plasma EV and pEV levels were increased in sPE when compared to normotensive without a significant size distribution difference in sPE (108.8 ± 30.2 nm) and normotensive-EVs (101.3 ± 20.3 nm). Impaired endothelial repair and proliferation, reduced endothelial barrier function, reduced endothelial-dependent vasorelaxation, and decreased nitrite level indicate that sPE-EVs induced vascular endothelial dysfunction. Moreover, sPE-EVs significantly downregulated endothelial nitric oxide synthase (eNOS and p-eNOS) when compared to normotensive-EV. CONCLUSIONS: EVs from sPE women impair endothelial-dependent vascular functions in vitro.

Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic ß2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.

Acetate, a Short-Chain Fatty Acid, Acutely Lowers Heart Rate and Cardiac Contractility Along with Blood Pressure.

Short-chain fatty acids (SCFAs) are metabolites produced almost exclusively by the gut microbiota and are an essential mechanism by which gut microbes influence host physiology. Given that SCFAs induce vasodilation, we hypothesized that they might have additional cardiovascular effects. In this study, novel mechanisms of SCFA action were uncovered by examining the acute effects of SCFAs on cardiovascular physiology in vivo and ex vivo. Acute delivery of SCFAs in conscious radiotelemetry-implanted mice results in a simultaneous decrease in both mean arterial pressure and heart rate (HR). Inhibition of sympathetic tone by the selective ß-1 adrenergic receptor antagonist atenolol blocks the acute drop in HR seen with acetate administration, yet the decrease in mean arterial pressure persists. Treatment with tyramine, an indirect sympathomimetic, also blocks the acetate-induced acute drop in HR. Langendorff preparations show that acetate lowers HR only after long-term exposure and at a smaller magnitude than seen in vivo. Pressure-volume loops after acetate injection show a decrease in load-independent measures of cardiac contractility. Isolated trabecular muscle preparations also show a reduction in force generation upon SCFA treatment, though only at supraphysiological concentrations. These experiments demonstrate a direct cardiac component of the SCFA cardiovascular response. These data show that acetate affects blood pressure and cardiac function through parallel mechanisms and establish a role for SCFAs in modulating sympathetic tone and cardiac contractility, further advancing our understanding of the role of SCFAs in blood pressure regulation. SIGNIFICANCE STATEMENT: Acetate, a short-chain fatty acid, acutely lowers heart rate (HR) as well as mean arterial pressure in vivo in radiotelemetry-implanted mice. Acetate is acting in a sympatholytic manner on HR and exerts negative inotropic effects in vivo. This work has implications for potential short-chain fatty acid therapeutics as well as gut dysbiosis-related disease states.

Aging and chronic high-fat feeding negatively affect kidney size, function, and gene expression in CTRP1-deficient mice.

C1q/TNF-related protein 1 (CTRP1) is an endocrine factor with metabolic, cardiovascular, and renal functions. We previously showed that aged Ctrp1-knockout (KO) mice fed a control low-fat diet develop renal hypertrophy and dysfunction. Since aging and obesity adversely affect various organ systems, we hypothesized that aging, in combination with obesity induced by chronic high-fat feeding, would further exacerbate renal dysfunction in CTRP1-deficient animals. To test this, we fed wild-type and Ctrp1-KO mice a high-fat diet for 8 mo or longer. Contrary to our expectation, no differences were observed in blood pressure, heart function, or vascular stiffness between genotypes. Loss of CTRP1, however, resulted in an approximately twofold renal enlargement (relative to body weight), ∼60% increase in urinary total protein content, and elevated pH, and changes in renal gene expression affecting metabolism, signaling, transcription, cell adhesion, solute and metabolite transport, and inflammation. Assessment of glomerular integrity, the extent of podocyte foot process effacement, as well as renal response to water restriction and salt loading did not reveal significant differences between genotypes. Interestingly, blood platelet, white blood cell, neutrophil, lymphocyte, and eosinophil counts were significantly elevated, whereas mean corpuscular volume and hemoglobin were reduced in Ctrp1-KO mice. Cytokine profiling revealed increased circulating levels of CCL17 and TIMP-1 in KO mice. Compared with our previous study, current data suggest that chronic high-fat feeding affects renal phenotypes differently than similarly aged mice fed a control low-fat diet, highlighting a diet-dependent contribution of CTRP1 deficiency to age-related changes in renal structure and function.

Late-onset renal hypertrophy and dysfunction in mice lacking CTRP1.

Local and systemic factors that influence renal structure and function in aging are not well understood. The secretory protein C1q/TNF-related protein 1 (CTRP1) regulates systemic metabolism and cardiovascular function. We provide evidence here that CTRP1 also modulates renal physiology in an age- and sex-dependent manner. In mice lacking CTRP1, we observed significantly increased kidney weight and glomerular hypertrophy in aged male but not female or young mice. Although glomerular filtration rate, plasma renin and aldosterone levels, and renal response to water restriction did not differ between genotypes, CTRP1-deficient male mice had elevated blood pressure. Echocardiogram and pulse wave velocity measurements indicated normal heart function and vascular stiffness in CTRP1-deficient animals, and increased blood pressure was not due to greater salt retention. Paradoxically, CTRP1-deficient mice had elevated urinary sodium and potassium excretion, partially resulting from reduced expression of genes involved in renal sodium and potassium reabsorption. Despite renal hypertrophy, markers of inflammation, fibrosis, and oxidative stress were reduced in CTRP1-deficient mice. RNA sequencing revealed alterations and enrichments of genes in metabolic processes in CTRP1-deficient animals. These results highlight novel contributions of CTRP1 to aging-associated changes in renal physiology.

Novel Expression of GABAA Receptors on Resistance Arteries That Modulate Myogenic Tone.

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries.

Airway smooth muscle photorelaxation via opsin receptor activation.

Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.

Deletion of the microRNA-degrading nuclease, translin/trax, prevents pathogenic vascular stiffness.

Vascular stiffness plays a key role in the pathogenesis of hypertension. Recent studies indicate that the age-associated reduction in miR-181b levels in vascular smooth muscle cells (VSMCs) contributes to increased vascular stiffness. As these findings suggest that inhibiting degradation of miR-181b might prevent vascular stiffening, we have assessed whether the microRNA-degrading translin/trax (TN/TX) complex mediates degradation of miR-181b in the aorta.We found that TN-/- mice display elevated levels of miR-181b expression in the aorta. Therefore, we tested whether TN deletion prevents vascular stiffening in a mouse model of hypertension, induced by chronic high-salt intake (4%NaCl in drinking water for 3 wk; HSW). TN-/- mice subjected to HSW stress do not show increased vascular stiffness, as monitored by pulse wave velocity and tensile testing. The protective effect of TN deletion in the HSW paradigm appears to be mediated by its ability to increase miR-181b in the aorta since HSW decreases levels of miR-181b in WT mice, but not in TN KO mice. We demonstrate for the first time that interfering with microRNA degradation can have a beneficial impact on the vascular system and identify the microRNA-degrading TN/TX RNase complex as a potential therapeutic target in combatting vascular stiffness.NEW & NOTEWORTHY While the biogenesis and mechanism of action of mature microRNA are well understood, much less is known about the regulation of microRNA via degradation. Recent studies have identified the protein complex, translin(TN)/trax(TX), as a microRNA-degrading enzyme. Here, we demonstrate that TN/TX is expressed in vascular smooth muscle cells. Additionally, deletion of the TN/TX complex selectively increases aortic miR-181b and prevents increased vascular stiffness caused by ingestion of high-salt water. To our knowledge, this is first report describing the role of a microRNA RNAse in cardiovascular biology or pathobiology.

Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening.

Vascular stiffening and its sequelae are major causes of morbidity and mortality in the elderly. The increasingly accepted concept of "smooth muscle cell (SMC) stiffness syndrome" along with matrix deposition has emerged in vascular biology to account for the mechanical phenotype of arterial aging, but the molecular targets remain elusive. In this study, using an unbiased proteomic analysis, we identified lysyl oxidase-like 2 (LOXL2) as a critical SMC mediator for age-associated vascular stiffening. We tested the hypothesis that loss of LOXL2 function is protective in aging-associated vascular stiffening. We determined that exogenous and endogenous nitric oxide markedly decreased LOXL2 abundance and activity in the extracellular matrix of isolated SMCs and LOXL2 endothelial cells suppress LOXL2 abundance in the aorta. In a longitudinal study, LOXL2+/- mice were protected from age-associated increase in pulse-wave velocity, an index of vascular stiffening, as occurred in littermate wild-type mice. Using isolated aortic segments, we found that LOXL2 mediates vascular stiffening in aging by promoting SMC stiffness, augmented SMC contractility, and vascular matrix deposition. Together, these studies establish LOXL2 as a nodal point for a new therapeutic approach to treat age-associated vascular stiffening. NEW & NOTEWORTHY Increased central vascular stiffness augments risk of major adverse cardiovascular events. Despite significant advances in understanding the genetic and molecular underpinnings of vascular stiffening, targeted therapy has remained elusive. Here, we show that lysyl oxidase-like 2 (LOXL2) drives vascular stiffening during aging by promoting matrix remodeling and vascular smooth muscle cell stiffening. Reduced LOXL2 expression protects mice from age-associated vascular stiffening and delays the onset of isolated systolic hypertension, a major consequence of stiffening.

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.

Pulse wave travel distance as a novel marker of ventricular-arterial coupling.

Each stroke volume ejected by the heart is distributed along the arterial system as a pressure waveform. How far the front of the pressure waveform travels within the arterial system depends both on the pulse wave velocity (PWV) and the ejection time (ET). We tested the hypothesis that ET and PWV are coupled together, in order to produce a pulse wave travel distance (PWTD = PWV × ET) which would match the distance from the heart to the most distant site in the arterial system. The study was conducted in 11 healthy volunteers. We recorded lead II of the ECG along with pulse plethysmography at ear, finger and toe. The ET at the ear and pulse arrival time to each peripheral site were extracted. We then calculated PWV followed by PWTD for each location. Taken into account the individual subject variability PWTDToe in the supine position was 153 cm (95% CI 146-160 cm). It was not different from arterial pathway distance from the heart to the toe (D Toe 153 cm). The PWTDFinger and PWTDEar were longer than the distance from the heart to the finger and ear irrespective of body position. ETEar and PWVToe appear to be coupled in healthy subjects to produce a PWTD that is roughly equivalent to the arterial pathway distance to the toe. We propose that PWTD should be evaluated further to test its potential as a noninvasive parameter of ventricular-arterial coupling in subjects with cardiovascular diseases.

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.

Role of tissue transglutaminase in age-associated ventricular stiffness.

Aging is associated with increased cardiomyocyte loss, left-ventricular hypertrophy, and the accumulation of extracellular matrix, which results in declining cardiac function. The role of the matrix crosslinking enzyme, tissue transglutaminase (TG2), in age-related myocardial stiffness, and contractile function remains incompletely understood. In this study, we examined the role of TG2 in cardiac function, and determined whether TG2 inhibition can prevent age-associated changes in cardiac function. Male Fisher rats (18-month-old) were administered the transglutaminase inhibitor cystamine (study group) or saline (age-matched controls) for 12 weeks via osmotic mini-pumps. Cardiac function was determined by echocardiography and invasive pressure-volume loops. Rat hearts were dissected out, and TG2 expression, activity, and S-nitrosation were determined. Young (6-month-old) males were used as controls. TG2 activity significantly increased in the saline-treated but not in the cystamine-treated aging rat hearts. TG2 expression also increased with age and was unaltered by cystamine treatment. Aged rats showed increased left ventricular (LV) end-systolic dimension and a decrease in fractional shortening compared with young, which was not affected by cystamine. However, cystamine treatment preserved the preload-independent index of LV filling pressure and restored end-diastolic pressure, end-diastolic pressure-volume relationships, and arterial elastance toward young. An increase in TG2 activity contributes to age-associated increase in diastolic stiffness, thereby contributing to age-associated diastolic dysfunction. TG2 may thus represent a novel target for age-associated diastolic heart failure.