The FXYD1 protein plays a protective role against pulmonary hypertension and arterial remodeling via redox and inflammatory mechanisms.

Pulmonary hypertension (PH) consists of a heterogenous group of diseases that culminate in increased pulmonary arterial pressure and right ventricular (RV) dysfunction. We sought to investigate the role of FXYD1, a small membrane protein that modulates Na+-K+-ATPase function, in the pathophysiology of PH. We mined online transcriptome databases to assess FXYD1 expression in PH. We characterized the effects of FXYD1 knockout (KO) in mice on right and left ventricular (RV and LV) function using echocardiography and measured invasive hemodynamic measurements under normal conditions and after treatment with bleomycin sulfate or chronic hypoxia to induce PH. Using immunohistochemistry, immunoblotting, and functional assays, we examined the effects of FXYD1 KO on pulmonary microvasculature and RV and LV structure and assessed signaling via endothelial nitric oxide synthase (eNOS) and inflammatory pathways. FXYD1 lung expression tended to be lower in samples from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with controls, supporting a potential pathophysiological role. FXYD1 KO mice displayed characteristics of PH including significant increases in pulmonary arterial pressure, increased muscularization of small pulmonary arterioles, and impaired RV systolic function, in addition to LV systolic dysfunction. However, when PH was stimulated with standard models of lung injury-induced PH, there was no exacerbation of disease in FXYD1 KO mice. Both the lungs and left ventricles exhibited elevated nitrosative stress and inflammatory milieu. The absence of FXYD1 in mice results in LV inflammation and cardiopulmonary redox signaling changes that predispose to pathophysiological features of PH, suggesting FXYD1 may be protective.NEW & NOTEWORTHY This is the first study to show that deficiency of the FXYD1 protein is associated with pulmonary hypertension. FXYD1 expression is lower in the lungs of people with idiopathic pulmonary artery hypertension. FXYD1 deficiency results in both left and right ventricular functional impairment. Finally, FXYD1 may endogenously protect the heart from oxidative and inflammatory injury.

An update on emerging drugs for the treatment of hypercholesterolemia.

INTRODUCTION: Elevated levels of low-density lipoprotein (LDL) cholesterol have been unequivocally demonstrated to play a causal role in atherosclerotic cardiovascular disease. The last thirty years have witnessed a generation of clinical trials that have demonstrated a reduction in cardiovascular risk with the use of increasing intensive lipid lowering regimens involving statin therapy in combination with other agents. However, many patients fail to achieve treatment mandated LDL cholesterol goals. This highlights the need to develop additional approaches to lower LDL cholesterol levels. AREAS COVERED: (i) Contemporary data highlighting the atherogenicity of LDL cholesterol and cardiovascular benefits of current lipid lowering therapies. (ii) Importance of statin intolerance and inability to achieve LDL cholesterol goals in driving ongoing cardiovascular risk. (iii) Emergence of new therapeutic agents designed to achieve more effective lowering of LDL cholesterol. EXPERT OPINION: Effective lowering of LDL cholesterol plays a critical role in approaches to the prevention of cardiovascular disease. A greater number of patients will require combinations of agents to achieve optimal lipid control. Accordingly, new agents will be required to provide sufficient choice for patients at high cardiovascular risk.

Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure.

Heart failure (HF) is a shared manifestation of several cardiovascular pathologies, including hypertension and myocardial infarction, and a limited repertoire of treatment modalities entails that the associated morbidity and mortality remain high. Impaired nitric oxide (NO)/guanylyl cyclase (GC)/cyclic guanosine-3',5'-monophosphate (cGMP) signaling, underpinned, in part, by up-regulation of cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) isozymes, contributes to the pathogenesis of HF, and interventions targeted to enhancing cGMP have proven effective in preclinical models and patients. Numerous PDE isozymes coordinate the regulation of cardiac cGMP in the context of HF; PDE2 expression and activity are up-regulated in experimental and human HF, but a well-defined role for this isoform in pathogenesis has yet to be established, certainly in terms of cGMP signaling. Herein, using a selective pharmacological inhibitor of PDE2, BAY 60-7550, and transgenic mice lacking either NO-sensitive GC-1α (GC-1α-/-) or natriuretic peptide-responsive GC-A (GC-A-/-), we demonstrate that the blockade of PDE2 promotes cGMP signaling to offset the pathogenesis of experimental HF (induced by pressure overload or sympathetic hyperactivation), reversing the development of left ventricular hypertrophy, compromised contractility, and cardiac fibrosis. Moreover, we show that this beneficial pharmacodynamic profile is maintained in GC-A-/- mice but is absent in animals null for GC-1α or treated with a NO synthase inhibitor, revealing that PDE2 inhibition preferentially enhances NO/GC/cGMP signaling in the setting of HF to exert wide-ranging protection to preserve cardiac structure and function. These data substantiate the targeting of PDE2 in HF as a tangible approach to maximize myocardial cGMP signaling and enhancing therapy.

The Riskier Lipid: What Is on the HORIZON for Lipoprotein (a) and Should There Be Lp(a) Screening for All?

PURPOSE OF REVIEW: Despite widespread targeting of cardiovascular risk factors, many patients continue to experience clinical events. This residual risk has stimulated efforts to develop novel therapeutic approaches to target additional factors underscoring cardiovascular disease. This review aimed to summarize existing evidence supporting targeting of Lp(a) as a novel cardioprotective strategy. RECENT FINDINGS: Increasing evidence has implicated lipoprotein (a) [Lp(a)] in the pathogenesis of both atherosclerotic and calcific aortic valve disease. Therapeutic advances have produced novel agents that selectively lower Lp(a) levels, which have now progressed to evaluate their impact on cardiovascular events in large clinical outcome trials. Evidence continues to accumulate suggesting that targeting Lp(a) may be effective in reducing cardiovascular risk. With advances in Lp(a) targeted therapeutics, clinical trials now have the opportunity to determine whether this strategy will be effective for high-risk patients.

Endothelial C-Type Natriuretic Peptide Is a Critical Regulator of Angiogenesis and Vascular Remodeling.

BACKGROUND: Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia. METHODS: The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP-/-, global natriuretic peptide receptor (NPR)-B-/- and NPR-C-/- animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease. RESULTS: Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP-/- and NPR-C-/-, but not NPR-B-/-, mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of Gi, ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level. CONCLUSIONS: These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.

The novel P2X7 receptor antagonist PKT100 improves cardiac function and survival in pulmonary hypertension by direct targeting of the right ventricle.

In pulmonary hypertension (PH) a proinflammatory milieu drives pulmonary vascular remodeling, maladaptive right ventricular (RV) remodeling, and right-sided heart failure. There is an unmet need for RV-targeted pharmaco-therapies to improve mortality. Targeting of the P2X7 receptor (P2X7R) reduces pulmonary pressures; however, its effects on the RV are presently unknown. We investigated the effect of P2X7 receptor (P2X7R) inhibition on the pulmonary vasculature and RV remodeling using the novel P2X7R antagonist PKT100. C57BL/6 mice were administered intratracheal bleomycin or saline and treated with PKT100 (0.2 mg·kg-1·day-1) or DMSO vehicle. RV was assessed by right heart catheterization and echocardiography, 21 days posttreatment. Cytokines in serum and bronchoalveolar lavage fluid (BALF) were analyzed by ELISA and flow cytometry. Lungs and hearts were analyzed histologically for pulmonary vascular and RV remodeling. Focused-PCR using genes involved in RV remodeling was performed. Right ventricular systolic pressure (RVSP) was elevated in bleomycin-treated mice (30.2 ± 1.1; n = 7) compared with control mice (23.5 ± 1.0; n = 10; P = 0.008). PKT100 treatment did not alter RVSP (32.4 ± 1.8; n = 9), but it substantially improved survival (93% vs. 57% DMSO). There were no differences between DMSO and PKT100 bleomycin mice in pulmonary inflammation or remodeling. However, RV hypertrophy was reduced in PKT100 mice. Bleomycin decreased echocardiographic surrogates of RV systolic performance, which were significantly improved with PKT100. Four genes involved in RV remodeling (RPSA, Rplp0, Add2, and Scn7a) were differentially expressed between DMSO and PKT100-treated groups. The novel P2X7R inhibitor, PKT100, attenuates RV hypertrophy and improves RV contractile function and survival in a mouse model of PH independently of effects on the pulmonary vasculature. PKT100 may improve ventricular response to increased afterload and merits further investigation into the potential role of P2X7R antagonists as direct RV-focused therapies in PH.NEW & NOTEWORTHY This study demonstrates the therapeutic potential for right-sided heart failure of a novel inhibitor of the P2X7 receptor (P2X7R). Inflammatory signaling and right ventricular function were improved in a mouse model of pulmonary fibrosis with secondary pulmonary hypertension when treated with this inhibitor. Importantly, survival was also improved, suggesting that this inhibitor, and other P2X7R antagonists, could be uniquely effective in right ventricle (RV)-targeted therapy in pulmonary hypertension. This addresses a major limitation of current treatment options, where the significant improvements in pulmonary pressures ultimately do not prevent mortality due to RV failure.

Modified redox signaling in vasculature after chronic infusion of the insulin receptor antagonist, S961.

BACKGROUND: Type 2 diabetes and associated vascular complications cause substantial morbidity and mortality. It is important to investigate mechanisms and test therapies in relevant physiological models, yet few animal models adequately recapitulate all aspects of the human condition. OBJECTIVE: We sought to determine the potential of using an insulin receptor antagonist, S961, in mice for investigating vascular pathophysiology. METHODS: S961 was infused into mice for 4 weeks. Blood glucose was monitored, and insulin was measured at the end of the protocol. Blood pressure and pressor responses to vasodilators were measured in cannulated mice, and vascular reactive oxygen and nitrogen species were measured in isolated tissue. RESULTS: S961 infusion-induced hyperglycemia and hyperinsulinemia. There was evidence of increased vascular reactive oxygen and nitrogen species and modification of NO-mediated signaling. Pressor responses to a NO donor were attenuated, but responses to bradykinin were preserved. CONCLUSIONS: Infusion of S961, an insulin receptor antagonist, results in the production of a mouse model of type 2 diabetes that may be useful for investigating redox signaling in the vasculature of insulin-resistant mice over the short term. It is limited by both the transient nature of the hyperglycemia and incomplete functional analogy to the human condition.

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis.

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFß1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFß1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFß1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFß1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

Inhibition of phosphodiesterase 2 augments cGMP and cAMP signaling to ameliorate pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin contributes to PH pathogenesis, and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP, which underpin the bioactivity of NO and prostacyclin. PDE5 inhibitors (eg, sildenafil) are licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH. METHODS AND RESULTS: The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide- and treprostinil-evoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), trepostinil, inorganic nitrate (NO donor), or a PDE5 inhibitor. Proliferation of pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension was reduced by BAY 60-7550, an effect further enhanced in the presence of atrial natriuretic peptide, NO, and treprostinil. CONCLUSIONS: PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity and is additive with prostacyclin analogues, PDE5 inhibitor, and NO. PDE2 inhibition represents a viable, orally active therapy for PH.

Accelerated age-related decline in renal and vascular function in female rats following early-life growth restriction.

Many studies report sexual dimorphism in the fetal programming of adult disease. We hypothesized that there would be differences in the age-related decline in renal function between male and female intrauterine growth-restricted rats. Early-life growth restriction was induced in rat offspring by administering a low-protein diet (LPD; 8.7% casein) to dams during pregnancy and lactation. Control dams were fed a normal-protein diet (NPD; 20% casein). Mean arterial pressure (MAP) and renal structure and function were assessed in 32- and 100-wk-old offspring. Mesenteric artery function was examined at 100 wk using myography. At 3 days of age, body weight was ∼24% lower (P < 0.0001) in LPD offspring; this difference was still apparent at 32 wk but not at 100 wk of age. MAP was not different between the male NPD and LPD groups at either age. However, MAP was greater in LPD females compared with NPD females at 100 wk of age (∼10 mmHg; P < 0.001). Glomerular filtration rate declined with age in the NPD male, LPD male and LPD female offspring (∼45%, all P < 0.05), but not in NPD female offspring. Mesenteric arteries in the aged LPD females had reduced sensitivity to nitric oxide donors compared with their NPD counterparts, suggesting that vascular dysfunction may contribute to the increased risk of disease in aged females. In conclusion, females growth-restricted in early life were no longer protected from an age-related decline in renal and arterial function, and this was associated with increased arterial pressure without evidence of renal structural damage.

Exploring the Therapeutic Potential of C-Type Natriuretic Peptide for Preeclampsia.

BACKGROUND: Preeclampsia is a serious condition of pregnancy, complicated by aberrant maternal vascular dysfunction. CNP (C-type natriuretic peptide) contributes to vascular homeostasis, acting through NPR-B (natriuretic peptide receptor-B) and NPR-C (natriuretic peptide receptor-C). CNP mitigates vascular dysfunction of arteries in nonpregnant cohorts; this study investigates whether CNP can dilate maternal arteries in ex vivo preeclampsia models. METHODS: Human omental arteries were dissected from fat biopsies collected during cesarean section. CNP, NPR-B, and NPR-C mRNA expression was assessed in arteries collected from pregnancies complicated by preeclampsia (n=6) and normotensive controls (n=11). Using wire myography, we investigated the effects of CNP on dilation of arteries from normotensive pregnancies. Arteries were preconstricted with either serum from patients with preeclampsia (n=6) or recombinant ET-1 (endothelin-1; vasoconstrictor elevated in preeclampsia; n=6) to model vasoconstriction associated with preeclampsia. Preconstricted arteries were treated with recombinant CNP (0.001-100 µmol/L) or vehicle and vascular relaxation assessed. In further studies, arteries were preincubated with NPR-B (5 µmol/L) and NPR-C (10 µmol/L) antagonists before serum-induced constriction (n=4-5) to explore mechanistic signaling. RESULTS: CNP, NPR-B, and NPR-C mRNAs were not differentially expressed in omental arteries from preeclamptic pregnancies. CNP potently stimulated maternal artery vasorelaxation in our model of preeclampsia (using preeclamptic serum). Its vasodilatory actions were driven through the activation of NPR-B predominantly; antagonism of this receptor alone dampened CNP vasorelaxation. Interestingly, CNP did not reduce ET-1-driven omental artery constriction. CONCLUSIONS: Collectively, these data suggest that enhancing CNP signaling through NPR-B offers a potential therapeutic strategy to reduce systemic vascular constriction in preeclampsia.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a novel activator of transient receptor potential vanilloid 1 (TRPV1) channel.

TRPV1 is a member of the transient receptor potential ion channel family and is gated by capsaicin, the pungent component of chili pepper. It is expressed predominantly in small diameter peripheral nerve fibers and is activated by noxious temperatures >42 °C. 20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P-450 4A/4F-derived metabolite of the membrane phospholipid arachidonic acid. It is a powerful vasoconstrictor and has structural similarities with other TRPV1 agonists, e.g. the hydroperoxyeicosatetraenoic acid 12-HPETE, and we hypothesized that it may be an endogenous ligand for TRPV1 in sensory neurons innervating the vasculature. Here, we demonstrate that 20-HETE both activates and sensitizes mouse and human TRPV1, in a kinase-dependent manner, involving the residue Ser(502) in heterologously expressed hTRPV1, at physiologically relevant concentrations.

Impact of lipid lowering on coronary atherosclerosis moving from the lumen to the artery wall.

Randomized clinical trials have demonstrated that increasingly intensive lowering of low-density lipoprotein cholesterol (LDL-C) reduces the rate of cardiovascular events in the primary and secondary prevention setting. Integration of serial coronary imaging within clinical trials has enabled evaluation of medical therapies on the natural history of coronary disease. These studies have extended from early investigation of coronary obstruction with angiography to more contemporary evaluation of plaque burden and composition with imaging modalities that directly visualize the artery wall. The findings of these trials have demonstrated that intensive lipid lowering promotes plaque regression and stabilization. The lessons of this body of research provide a biological rationale underscoring the ability of intensive lipid lowering to reduce cardiovascular risk and have the potential to promote greater uptake in clinical practice.

Targeting triglycerides to lower residual cardiovascular risk.

INTRODUCTION: Dyslipidemia therapeutics have primarily focused on lowering levels of low-density lipoprotein cholesterol. However, many patients continue to experience cardiovascular events, despite effective lowering of LDL-C. This has prompted efforts to target additional risk factors to achieve more effective prevention of cardiovascular disease. Emerging evidence suggests that triglyceride rich lipoproteins play a causal role in atherosclerosis, highlighting the potential for specific therapeutic lowering. AREAS COVERED: (1) Evidence to support the causal role of triglyceride rich lipoproteins in atherosclerotic cardiovascular disease. (2) Use of existing lipid modifying therapies to target triglyceride rich lipoproteins. (3) Development of novel therapeutic agents that target triglyceride rich lipoproteins and their potential impact on cardiovascular risk. EXPERT OPINION/COMMENTARY: Evidence from preclinical, observational and genetic studies highlight the role of triglyceride rich lipoproteins in the causal pathway of atherosclerotic cardiovascular disease. A number of existing agents have the potential to reduce residual cardiovascular risk associated with hypertriglyceridemia. However, emerging agents have the potential to substantially and preferentially lower triglyceride levels beyond contemporary therapeutics. How they will modulate cardiovascular risk will ultimately be determined by large clinical outcomes trials. They do provide the opportunity to substantially influence the way we target dyslipidemia in the prevention of cardiovascular disease.

Impact of reduced uterine perfusion pressure model of preeclampsia on metabolism of placenta, maternal and fetal hearts.

Preeclampsia is a cardiovascular pregnancy complication characterised by new onset hypertension and organ damage or intrauterine growth restriction. It is one of the leading causes of maternal and fetal mortality in pregnancy globally. Short of pre-term delivery of the fetus and placenta, treatment options are limited. Consequently, preeclampsia leads to increased cardiovascular disease risk in both mothers and offspring later in life. Here we aim to examine the impact of the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia on the maternal cardiovascular system, placental and fetal heart metabolism. The surgical RUPP model was induced in pregnant rats by applying silver clips around the aorta and uterine arteries on gestational day 14, resulting in ~ 40% uterine blood flow reduction. The experiment was terminated on gestational day 19 and metabolomic profile of placentae, maternal and fetal hearts analysed using high-resolution 1H NMR spectroscopy. Impairment of uterine perfusion in RUPP rats caused placental and cardiac hypoxia and a series of metabolic adaptations: altered energetics, carbohydrate, lipid and amino acid metabolism of placentae and maternal hearts. Comparatively, the fetal metabolic phenotype was mildly affected. Nevertheless, long-term effects of these changes in both mothers and the offspring should be investigated further in the future.

High-Resolution Transthoracic Echocardiography Accurately Detects Pulmonary Arterial Pressure and Decreased Right Ventricular Contractility in a Mouse Model of Pulmonary Fibrosis and Secondary Pulmonary Hypertension.

Background To date, assessment of right ventricular (RV) function in mice has relied extensively on invasive measurements. Echocardiographic advances have allowed adaptation of measures used in humans for serial, noninvasive RV functional assessment in mice. We evaluated the diagnostic performance of tricuspid annular plane systolic excursion (TAPSE), RV peak systolic myocardial velocity (s'), RV myocardial performance index (MPI), and RV fractional area change (FAC) in a mouse model of pulmonary hypertension. Methods and Results Echocardiography was performed on mice at baseline and 3 weeks after induction of pulmonary hypertension using inhaled bleomycin or saline, including adapted measures of TAPSE, s', MPI, and FAC. RV systolic pressure was measured by invasive catheterization, and RV contractility was measured as the peak slope of the RV systolic pressure recording (maximum change pressure/change time). Postmortem morphological assessment of RV hypertrophy was performed. RV systolic pressure was elevated and maximum change pressure/change time was reduced in bleomycin versus control (n=8; P=0.002). Compared with controls, bleomycin mice had reduced TAPSE (0.79±0.05 versus 1.06±0.04 mm; P=0.003), s' (21.3±1.2 versus 29.2±1.3 mm/s; P<0.001), and FAC (20.3±0.7% versus 31.0±1.3%; P<0.001), whereas MPI was increased (0.51±0.03 versus 0.37±0.01; P=0.006). All measures correlated with RV systolic pressure and maximum change pressure/change time. Intraobserver and interobserver variability were minimal. Receiver operating characteristic curves demonstrated that TAPSE (<0.84 mm), s'(<23.3 mm/s), MPI (0.42), and FAC (<23.3%) identified maximum change pressure/change time ≤2100 mm Hg/s with high accuracy. Conclusions TAPSE, s', MPI, and FAC are measurable consistently using high-resolution echocardiography in mice, and are sensitive and specific measures of pulmonary pressure and RV function. This validation opens the opportunity for serial noninvasive measures in mouse models of pulmonary hypertension, enhancing the statistical power of preclinical studies of novel therapeutics.

20-hydroxyeicosatetraenoic acid (20-HETE) is a pivotal endogenous ligand for TRPV1-mediated neurogenic inflammation in the skin.

BACKGROUND AND PURPOSE: Transient receptor potential cation channel subfamily V member 1 (TRPV1) is localized to sensory C-fibres and its opening leads to membrane depolarization, resulting in neuropeptide release and neurogenic inflammation. However, the identity of the endogenous activator of TRPV1 in this setting is unknown. The arachidonic acid metabolites 12-hydroperoxyeicosatetraenoyl acid (12-HpETE) and 20-hydroxyeicosatetraenoic acid (20-HETE) have emerged as potential endogenous activators of TRPV1. However, whether these lipids underlie TRPV1-mediated neurogenic inflammation remains unknown. EXPERIMENTAL APPROACH: We analysed human cantharidin-induced blister samples and inflammatory responses in TRPV1 transgenic mice. KEY RESULTS: In a human cantharidin-blister model, the potent TRPV1 activators 20-HETE but not 12-HETE (stable metabolite of 12-HpETE) correlated with arachidonic acid levels. Similarly, in mice, levels of 20-HETE (but not 12-HETE) and arachidonic acid were strongly positively correlated within the inflammatory milieu. Furthermore, LPS-induced oedema formation and neutrophil recruitment were substantially and significantly attenuated by pharmacological block or genetic deletion of TRPV1 channels, inhibition of 20-HETE formation or SP receptor neurokinin 1 (NK1 ) blockade. LPS treatment also increased cytochrome P450 ω-hydroxylase gene expression, the enzyme responsible for 20-HETE production. CONCLUSION AND IMPLICATIONS: Taken together, our findings suggest that endogenously generated 20-HETE activates TRPV1 causing C-fibre activation and consequent oedema formation. These findings identify a novel pathway that may be useful in the therapeutics of diseases/conditions characterized by a prominent neurogenic inflammation, as in several skin diseases.

FXYD1 Is Protective Against Vascular Dysfunction.

[Figure: see text].

ß 3 Adrenergic Receptor Stimulation Promotes Reperfusion in Ischemic Limbs in a Murine Diabetic Model.

Aims/Hypothesis: Peripheral arterial disease (PAD) is a major burden, resulting in limb claudication, repeated surgical interventions and amputation. There is an unmet need for improved medical management of PAD that improves quality of life, maintains activities of daily life and reduces complications. Nitric oxide (NO)/redox balance is a key regulator of angiogenesis. We have previously shown beneficial effects of a ß 3 adrenergic receptor (ß 3AR) agonist on NO/redox balance. We hypothesized that ß 3AR stimulation would have therapeutic potential in PAD by promoting limb angiogenesis. Methods: The effect of the ß 3AR agonist CL 316,243 (1-1,000 nmol/L in vitro, 1 mg/kg/day s. c) was tested in established angiogenesis assays with human endothelial cells and patient-derived endothelial colony forming cells. Post-ischemia reperfusion was determined in streptozotocin and/or high fat diet-induced diabetic and non-diabetic mice in vivo using the hind limb ischemia model. Results: CL 316,243 caused accelerated recovery from hind limb ischemia in non-diabetic and type 1 and 2 diabetic mice. Increased eNOS activity and decreased superoxide generation were detected in hind limb ischemia calf muscle from CL 316, 243 treated mice vs. controls. The protective effect of CL 316,243 in diabetic mice was associated with >50% decreases in eNOS glutathionylation and nitrotyrosine levels. The ß 3AR agonist directly promoted angiogenesis in endothelial cells in vitro. These pro-angiogenic effects were ß 3AR and NOS-dependent. Conclusion/Interpretation: ß 3AR stimulation increased angiogenesis in diabetic ischemic limbs, with demonstrable improvements in NO/redox balance and angiogenesis elicited by a selective agonist. The orally available ß 3AR agonist, Mirabegron, used for overactive bladder syndrome, makes translation to a clinical trial by repurposing of a ß 3AR agonist to target PAD immediately feasible.

Protocol for the Stimulating ß3-Adrenergic Receptors for Peripheral Artery Disease (STAR-PAD) trial: a double-blinded, randomised, placebo-controlled study evaluating the effects of mirabegron on functional performance in patients with peripheral arterial disease.

INTRODUCTION: There is currently only one approved medication effective at improving walking distance in people with intermittent claudication. Preclinical data suggest that the ß3-adrenergic receptor agonist (mirabegron) could be repurposed to treat intermittent claudication associated with peripheral artery disease. The aim of the Stimulating ß3-Adrenergic Receptors for Peripheral Artery Disease (STAR-PAD) trial is to test whether mirabegron improves walking distance in people with intermittent claudication. METHODS AND ANALYSIS: The STAR-PAD trial is a Phase II, multicentre, double-blind, randomised, placebo-controlled trial of mirabegron versus placebo on walking distance in patients with PAD. A total of 120 patients aged ≥40 years with stable PAD and intermittent claudication will be randomly assigned (1:1 ratio) to receive either mirabegron (50 mg orally once a day) or matched placebo, for 12 weeks. The primary endpoint is change in peak walking distance as assessed by a graded treadmill test. Secondary endpoints will include: (i) initial claudication distance; (ii) average daily step count and total step count and (iii) functional status and quality of life assessment. Mechanistic substudies will examine potential effects of mirabegron on vascular function, including brachial artery flow-mediate dilatation; MRI assessment of lower limb blood flow, tissue perfusion and arterial stiffness and numbers and angiogenesis potential of endothelial progenitor cells. Given that mirabegron is safe and clinically available for alternative purposes, a positive study is positioned to immediately impact patient care. ETHICS AND DISSEMINATION: The STAR-PAD trial is approved by the Northern Sydney Local Health District Human Research Ethics Committee (HREC/18/HAWKE/50). The study results will be published in peer-reviewed medical or scientific journals and presented at scientific meetings, regardless of the study outcomes. TRIAL REGISTRATION NUMBER: ACTRN12619000423112; Results.