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.

Sex differences and Role of Lysyl Oxidase Like 2 (LOXL2) in Angiotensin II-Induced Hypertension in Mice.

INTRODUCTION: Hypertension, a disease with known sexual dimorphism, accelerates aging associated arterial stiffening. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase like 2 (LOXL2) in hypertension induced arterial stiffening. METHODS: Hypertension was induced by Angiotensin II (AngII) infusion. Blood pressure and pulse wave velocity (PWV) were measured noninvasively. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. Uniaxial stretch of cultured cells was used to evaluate the effect of biomechanical strain. LOXL2's catalytic function was examined using knockout and inhibition. RESULTS: Ang II infusion induced hypertension in both genotypes and sexes. Hypertensive WT males had higher PWV and passive stiffness. Aortic remodeling with increased wall thickness, intralamellar distance, higher LOXL2, collagen I, and collagen IV content was noted in WT males. Females did not exhibit increased PWV. LOXL2-depletion improved aortic mechanics in both sexes. LOXL2-depletion improved hyper-contractility in males but not females. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in VSMCs. LOXL2's catalytic function facilitated VSMC alignment in response to biomechanical strain. CONCLUSION: In males, arterial stiffening in hypertension is driven by VSMC response and matrix remodeling; females are protected from stiffening independent of LOXL2. VSMCs are the primary source of LOXL2 in the aorta. Hypertension increases LOXL2 processing and collagen I accumulation in the aorta. Overall, LOXL2 depletion offers protection in young hypertensive males and females.

Lysyl oxidase like-2 in fibrosis and cardiovascular disease.

Fibrosis is an important and essential reparative response to injury that, if left uncontrolled, results in the excessive synthesis, deposition, remodeling, and stiffening of the extracellular matrix, which is deleterious to organ function. Thus, the sustained activation of enzymes that catalyze matrix remodeling and cross linking is a fundamental step in the pathology of fibrotic diseases. Recent studies have implicated the amine oxidase lysyl oxidase like-2 (LOXL2) in this process and established significantly elevated expression of LOXL2 as a key component of profibrotic conditions in several organ systems. Understanding the relationship between LOXL2 and fibrosis as well as the mechanisms behind these relationships can offer significant insights for developing novel therapies. Here, we summarize the key findings that demonstrate the link between LOXL2 and fibrosis and inflammation, examine current therapeutics targeting LOXL2 for the treatment of fibrosis, and discuss future directions for experiments and biomedical engineering.

Regenerative and durable small-diameter graft as an arterial conduit.

Despite significant research efforts, clinical practice for arterial bypass surgery has been stagnant, and engineered grafts continue to face postimplantation challenges. Here, we describe the development and application of a durable small-diameter vascular graft with tailored regenerative capacity. We fabricated small-diameter vascular grafts by electrospinning fibrin tubes and poly(ε-caprolactone) fibrous sheaths, which improved suture retention strength and enabled long-term survival. Using surface topography in a hollow fibrin microfiber tube, we enable immediate, controlled perfusion and formation of a confluent endothelium within 3-4 days in vitro with human endothelial colony-forming cells, but a stable endothelium is noticeable at 4 weeks in vivo. Implantation of acellular or endothelialized fibrin grafts with an external ultrathin poly(ε-caprolactone) sheath as an interposition graft in the abdominal aorta of a severe combined immunodeficient Beige mouse model supports normal blood flow and vessel patency for 24 weeks. Mechanical properties of the implanted grafts closely approximate the native abdominal aorta properties after just 1 week in vivo. Fibrin mediated cellular remodeling, stable tunica intima and media formation, and abundant matrix deposition with organized collagen layers and wavy elastin lamellae. Endothelialized grafts evidenced controlled healthy remodeling with delayed and reduced macrophage infiltration alongside neo vasa vasorum-like structure formation, reduced calcification, and accelerated tunica media formation. Our studies establish a small-diameter graft that is fabricated in less than 1 week, mediates neotissue formation and incorporation into the native tissue, and matches the native vessel size and mechanical properties, overcoming main challenges in arterial bypass surgery.

Oxidative Stress Contributes to Microvascular Endothelial Dysfunction in Men and Women With Major Depressive Disorder.

RATIONALE: In rodent models of depression, oxidative stress-induced reductions in NO bioavailability contribute to impaired endothelium-dependent dilation. Endothelial dysfunction is evident in major depressive disorder (MDD); however, the molecular mediators remain undefined. OBJECTIVE: We sought to translate preclinical findings to humans by testing the role of oxidative stress in mediating microvascular endothelial dysfunction, including potential modulatory influences of sex, in MDD. METHODS AND RESULTS: Twenty-four treatment-naive, otherwise healthy, young adults with MDD (14 women; 18-23 years) and 20 healthy adults (10 women; 19-30 years) participated. Red blood cell flux (laser Doppler flowmetry) was measured during graded intradermal microdialysis perfusion of the endothelium-dependent agonist acetylcholine, alone and in combination with an NO synthase inhibitor (L-NAME), a superoxide scavenger (Tempol), and an NADPH oxidase inhibitor (apocynin), as well as during perfusion of the endothelium-independent agonist sodium nitroprusside. Tissue oxidative stress markers (eg, nitrotyrosine abundance, superoxide production) were also quantified. Endothelium-dependent dilation was blunted in MDD and mediated by reductions in NO-dependent dilation. Endothelium-independent dilation was likewise attenuated in MDD. In MDD, there were no sex differences in either NO-mediated endothelium-dependent dilation or endothelium-independent dilation. Acute scavenging of superoxide or inhibition of NADPH oxidase improved NO-dependent dilation in MDD. Expression and activity of oxidative stress markers were increased in MDD. In a subset of adults with MDD treated with a selective serotonin reuptake inhibitor for their depressive symptoms and in remission (n=8; 7 women; 19-37 years), NO-mediated endothelium-dependent dilation was preserved, but endothelium-independent dilation was impaired, compared with healthy adults. CONCLUSIONS: Oxidative stress-induced reductions in NO-dependent dilation, as well as alterations in vascular smooth muscle function, directly contribute to microvascular dysfunction in MDD. Strategies targeting vascular oxidative stress may be viable therapeutic options for improving NO-mediated endothelial function and reducing cardiovascular risk in MDD.

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.

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.

Acute lysyl oxidase inhibition alters microvascular function in normotensive but not hypertensive men and women.

The lysyl oxidase (LOX) family of enzymes regulates collagen cross-linking. LOX is upregulated in hypertension, increasing vascular stiffness. In vivo human research is sparse, as long-term LOX inhibition in animals causes vascular instability. Our aim was to evaluate the effects of LOX inhibition on cutaneous microvascular function to determine whether LOX function was upregulated in hypertensive humans. Four intradermal microdialysis fibers were placed in the forearm of 10 young [age: 24 ± 1 yr, mean arterial pressure (MAP): 87 ± 2 mmHg], 10 normotensive (age: 50 ± 2 yr, MAP: 84 ± 1 mmHg), and 10 hypertensive (age: 53 ± 2 yr, MAP: 112 ± 2 mmHg) subjects. Two sites were perfused with 10 mM ß-aminopropionitrile (BAPN) to inhibit LOX. The remaining two sites were perfused with lactated Ringer solution (control). A norepinephrine dose response (10-12-10-2 M) was performed to examine receptor-mediated vasoconstrictor function. A sodium nitroprusside dose response (10-8-10-1.3 M) was performed to examine vascular smooth muscle vasodilator function. Red blood cell flux was measured via laser-Doppler flowmetry and normalized to cutaneous vascular conductance (flux/MAP). LogEC50 values were calculated to determine changes in vasosensitivity. Skin tissue samples were analyzed for both extracellular matrix-bound and soluble LOX. LOX inhibition augmented vasoconstrictor sensitivity in young (control: -6.0 and BAPN: -7.1, P = 0.03) and normotensive (control: -4.8 and BAPN: -7.0, P = 0.01) but not hypertensive (control: -6.0 and BAPN: -6.1, P = 0.79) men and women. Relative to young subjects, extracellular matrix-bound LOX expression was higher in hypertensive subjects (young: 100 ± 8 and hypertensive: 162 ± 8, P = 0.002). These results suggest that upregulated LOX may contribute to the vascular stiffness and microvascular dysfunction characteristic in hypertension. NEW & NOTEWORTHY Matrix-bound lysyl oxidase (LOX) and LOX-like 2 expression are upregulated in the microvasculature of hypertensive men and women. Microvascular responsiveness to exogenous stimuli is altered with localized LOX inhibition in healthy men and women but not hypertensive adults. The LOX family differentially affects microvascular function in hypertensive and normotensive men and women.

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.

Alterations in endothelin type B receptor contribute to microvascular dysfunction in women who have had preeclampsia.

Microvascular dysfunction originating during a preeclamptic pregnancy persists postpartum and probably contributes to increased CVD risk in these women. One putative mechanism contributing to this dysfunction is increased vasoconstrictor sensitivity to endothelin-1 (ET-1), mediated by alterations in ET-1 receptor type-B (ETBR). We evaluated ET-1 sensitivity, ETAR, and ETBR contributions to ET-1-mediated constriction, and the mechanistic role of ETBR in endothelium-dependent dilation in vivo in the microvasculature of postpartum women who had preeclampsia (PrEC, n=12) and control women who had a healthy pregnancy (HC, n=12). We hypothesized that (1) PrEC would have a greater vasoconstrictor response to ET-1, and (2) reduced ETBR-mediated dilation. We further hypothesized that ETBR-blockade would attenuate endothelium-dependent vasodilation in HC, but not PrEC. Microvascular reactivity was assessed by measurement of cutaneous vascular conductance responses to graded infusion of ET-1 (10-20-10-8 mol/l), ET-1 + 500 nmol/l BQ-123 (ETAR-blockade), and ET-1 + 300 nmol/l BQ-788 (ETBR-blockade), and during graded infusion of acetylcholine (ACh, 10-7-102 mmol/l) and a standardized local heating protocol with and without ETBR-inhibition. PrEC had an increased vasoconstriction response to ET-1 (P=0.02). PrEC demonstrated reduced dilation responses to selective ETBR stimulation with ET-1 (P=0.01). ETBR-inhibition augmented ET-1-mediated constriction in HC (P=0.01) but attenuated ET-1-mediated constriction in PrEC (P=0.003). ETBR-inhibition attenuated endothelium-dependent vasodilation responses to 100mmol/l ACh (P=0.04) and local heat (P=0.003) in HC but increased vasodilation (ACh: P=0.01; local heat: P=0.03) in PrEC. Women who have had preeclampsia demonstrate augmented vasoconstrictor sensitivity to ET-1, mediated by altered ETBR signaling. Furthermore, altered ETBR function contributes to diminished endothelium-dependent dilation in previously preeclamptic women.

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.

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.

Arginase Inhibition Reverses Endothelial Dysfunction, Pulmonary Hypertension, and Vascular Stiffness in Transgenic Sickle Cell Mice.

BACKGROUND: In sickle cell disease (SCD), hemolysis results in the release and activation of arginase, an enzyme that reciprocally regulates nitric oxide (NO) synthase activity and thus, NO production. Simply supplementing the common substrate L-arginine, however, fails to improve NO bioavailability. In this study, we tested the hypothesis that arginase inhibition would improve NO bioavailability and thereby attenuate systemic and pulmonary vascular endothelial dysfunction in transgenic mice with SCD. METHODS: We studied 5-month-old transgenic sickle cell (SC) mice and age matched wild-type (WT) controls. SC mice were treated with the arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH; approximately 400 µg/d) for 4 weeks or left untreated. RESULTS: Vascular arginase activity was significantly higher at baseline in untreated SC mice compared to WT controls (SC versus WT, 346 ± 69.3 vs 69 ± 17.3 pmol urea/mg protein/minute; P = 0.0043; n = 4-5 animals per group). Treatment with ABH may significantly decrease arginase activity to levels near WT controls (SC + ABH 125.2 ± 17.3 pmol urea/mg protein/minute; P = 0.0213). Aortic strips from untreated SC mice showed decreased NO and increased reactive oxygen species (ROS) production (NO: fluorescence rate 0.76 ± 0.14 vs 1.34 ± 0.17 RFU/s; P = 0.0005 and ROS: fluorescence rate 3.96 ± 1.70 vs 1.63 ± 1.20 RFU/s, P = 0.0039; n = 3- animals per group). SC animals treated with ABH for 4 weeks demonstrated NO (fluorescence rate: 1.16 ± 0.16) and ROS (fluorescence rate: 2.02 ± 0.45) levels comparable with age-matched WT controls (n = 3- animals per group). The maximal endothelial-dependent vasorelaxation response to acetylcholine was impaired in aortic rings from SC mice compared with WT (57.7% ± 8.4% vs 80.3% ± 11.0%; P = 0.02; n = 6 animals per group). The endothelial-independent response was not different between groups. In SC mice, the right ventricular cardiac output index and end-systolic elastance were similar (4.60 ± 0.51 vs 2.9 ± 0.85 mL/min/100 g and 0.89 ± 0.48 vs 0.58 ± 0.11 mm Hg/µL), whereas the pulmonary vascular resistance index and right ventricular end-systolic pressure were greater (2.9 ± 0.28 vs 5.5 ± 2.0 mm Hg × min/µL/100 g and 18.9 ± 1.1 vs 23.1 ± 4.0 mm Hg; n = 8 animals per group). Pulse wave velocity (a measure of arterial stiffness) was greater in SC mice compared with WT (3.74 ± 0.54 vs 3.25 ± 0.21 m/s; n = 20 animals per group), arginase inhibition for 4 weeks significantly reduced the vascular SC phenotype to one similar to WT animals (P = 0.0009). CONCLUSIONS: Arginase inhibition improves NO bioavailability and thereby attenuates systemic and pulmonary vascular endothelial dysfunction in transgenic mice with SCD. Therefore, arginase is a potential therapeutic target in the treatment of cardiovascular dysfunction in SCD.

Evidence for a functional vasodilatatory role for hydrogen sulphide in the human cutaneous microvasculature.

KEY POINTS: Hydrogen sulphide (H2 S) is vasoprotective, attenuates inflammation and modulates blood pressure in animal models; however, its specific mechanistic role in the human vasculature remains unclear. In the present study, we report the novel finding that the enzymes responsible for endogenous H2 S production, cystathionine-γ-lyase and 3-mercaptopyruvate sulphurtransferase, are expressed in the human cutaneous circulation. Functionally, we show that H2 S-induced cutaneous vasodilatation is mediated, in part, by tetraethylammonium-sensitive calcium-dependent potassium channels and not by ATP-sensitive potassium channels. In addition, nitric oxide and cyclo-oxygenase-derived byproducts are required for full expression of exogenous H2 S-mediated cutaneous vasodilatation. Future investigations of the potential role for H2 S with respect to modulating vascular function in humans may have important clinical implications for understanding the mechanisms underlying vascular dysfunction characteristic of multiple cardiovascular pathologies. ABSTRACT: The present study aimed to identify the presence of cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (3-MST), which endogenously produce hydrogen sulphide (H2 S), and to functionally examine the mechanisms of H2 S-induced vasodilatation in the human cutaneous microcirculation. CSE and 3-MST were quantified in forearm skin samples from 5 healthy adults (24 ± 3 years) using western blot analysis. For functional studies, microdialysis fibres were placed in the forearm skin of 12 healthy adults (25 ± 3 years) for graded infusions (0.01-100 mm) of sodium sulphide (Na2 S) and sodium hydrogen sulphide (NaHS). To define the mechanisms mediating H2 S-induced vasodilatation, microdialysis fibres were perfused with Ringer solution (control), a ATP-sensitive potassium channel (KATP ) inhibitor, an intermediate calcium-dependent potassium channel (KCa ) inhibitor, a non-specific KCa channel inhibitor or triple blockade. To determine the interaction of H2 S-mediated vasodilatation with nitric oxide (NO) and cyclo-oxygenase (COX) signalling pathways, microdialysis fibres were perfused with Ringer solution (control), a non-specific NO synthase inhibitor, a non-selective COX inhibitor or combined inhibition during perfusion of increasing doses of Na2 S. CSE and 3-MST were expressed in all skin samples. Na2 S and NaHS elicited dose-dependent vasodilatation. Non-specific KCa channel inhibition and triple blockade blunted Na2 S-induced vasodilatation (P < 0.05), whereas KATP and intermediate KCa channel inhibition had no effect (P > 0.05). Separate and combined inhibition of NO and COX attenuated H2 S-induced vasodilatation (all P < 0.05). CSE and 3-MST are expressed in the human microvasculature. Exogenous H2 S elicits cutaneous vasodilatation mediated by KCa channels and has a functional interaction with both NO and COX vasodilatatory signalling pathways.

Nitroprusside inhibits calcium-induced impairment of red blood cell deformability.

BACKGROUND: Red blood cell (RBC) deformation is critical for microvascular perfusion and oxygen delivery to tissues. Abnormalities in RBC deformability have been observed in aging, sickle cell disease, diabetes, and preeclampsia. Although nitric oxide (NO) prevents decreases in RBC deformability, the underlying mechanism is unknown. STUDY DESIGN AND METHODS: As an experimental model, we used ionophore A23187-mediated calcium influx in RBCs to reduce their deformability and investigated the role of NO donor sodium nitroprusside (SNP) and KCa3.1 (Gardos) channel blockers on RBC deformability (measured as elongation index [EI] by microfluidic ektacytometry). RBC intracellular Ca(2+) and extracellular K(+) were measured by inductively coupled plasma mass spectrometry and potassium ion selective electrode, respectively. RESULTS: SNP treatment of RBCs blocked the Ca(2+) (approx. 10 µmol/L)-induced decrease in RBC deformability (EI 0.34 ± 0.02 vs. 0.09 ± 0.01, control vs. Ca(2+) loaded, p < 0.001; and EI 0.37 ± 0.02 vs. 0.30 ± 0.01, SNP vs. SNP plus Ca(2+) loaded) as well as Ca(2+) influx and K(+) efflux. The SNP effect was similar to that observed after pharmacologic blockade of the KCa3.1 channel (with charybdotoxin or extracellular medium containing isotonic K(+) concentration). In RBCs from KCa3.1(-/-) mice, 10 µmol/L Ca(2+) loading did not decrease cellular deformability. A preliminary attempt to address the molecular mechanism of SNP protection suggests the involvement of cell surface thiols. CONCLUSION: Our results suggest that nitroprusside treatment of RBCs may protect them from intracellular calcium increase-mediated stiffness, which may occur during microvascular perfusion in diseased states, as well as during RBC storage.

ACE2 and TAS2R38 receptor expression in pediatric and adult patients in the nasal and oral cavity.

Objective: To investigate differences in angiotensin-converting-enzyme-2 (ACE2) and bitter taste receptor (TAS2R38) expression between patient age groups and comorbidities to characterize the pathophysiology of coronavirus 19(COVID-19) pandemic. ACE2 is the receptor implicated to facilitate SARS-CoV-2 infections and levels of expression may correlate to the severity of COVID-19 infection. TAS2R38 has many non-gustatory roles in disease, with some evidence of severe COVID-19 disease in certain receptor phenotypes. Methods: We conducted a prospective cohort study and collected nasal and lingual tissue from healthy pediatric (n = 22) and adult (n = 25) patients undergoing general anesthesia for elective procedures. RNA isolation and qPCR were performed with primers targeting ACE2 and TAS2R38. Results: A total of 25 adult (52% male; 44% obese) and 22 pediatric (50% male; 36% obese) patients were enrolled, pediatric tissue had 43% more nasal ACE2 RNA expression than adults with a median fold change of 0.69 (IQR 0.37, 0.98) in adults and 0.99 (IQR 0.74, 1.43) in children (p < .05). There were no differences between the age groups in ACE2 expression of lingual tissue (p = .14) or TAS2R38 expression collected from either nasal (p = 049) or lingual tissue (p = .49). Stratifying for obesity yielded similar differences between nasal ACE2 expression between adults and children with median fold change of 0.56 (IQR 0.32, 0.87) in adults and 1.0 (IQR 0.82, 1.52) in children (p < .05). Conclusions: ACE2 receptor expression is higher in nasal tissue collected from children compared to adults, suggesting COVID-19 infectivity is more complicated than ACE2 and TAS2R38 mRNA expression. Level of Evidence: NA.

An evolutionarily conserved olfactory receptor is required for sex differences in blood pressure.

Sex differences in blood pressure are well-established, with premenopausal women having lower blood pressure than men by ~10 millimeters of mercury; however, the underlying mechanisms are not fully understood. We report here that sex differences in blood pressure are absent in olfactory receptor 558 knockout (KO) mice. Olfr558 localizes to renin-positive cells in the kidney and to vascular smooth muscle cells. Female KOs exhibit increased blood pressure and increased pulse wave velocity. In contrast, male KO mice have decreased renin expression and activity, altered vascular reactivity, and decreased diastolic pressure. A rare OR51E1 (human ortholog) missense variant has a statistically significant sex interaction effect with diastolic blood pressure, increasing diastolic blood pressure in women but decreasing it in men. In summary, our findings demonstrate an evolutionarily conserved role for OLFR558/OR51E1 to mediate sex differences in blood pressure.