Effect of Coffee and Cocoa-Based Confectionery Containing Coffee on Markers of DNA Damage and Lipid Peroxidation Products: Results from a Human Intervention Study.

The effect of coffee and cocoa on oxidative damage to macromolecules has been investigated in several studies, often with controversial results. This study aimed to investigate the effect of one-month consumption of different doses of coffee or cocoa-based products containing coffee on markers of DNA damage and lipid peroxidation in young healthy volunteers. Twenty-one volunteers were randomly assigned into a three-arm, crossover, randomized trial. Subjects were assigned to consume one of the three following treatments: one cup of espresso coffee/day (1C), three cups of espresso coffee/day (3C), and one cup of espresso coffee plus two cocoa-based products containing coffee (PC) twice per day for 1 month. At the end of each treatment, blood samples were collected for the analysis of endogenous and H2O2-induced DNA damage and DNA oxidation catabolites, while urines were used for the analysis of oxylipins. On the whole, four DNA catabolites (cyclic guanosine monophosphate (cGMP), 8-OH-2'-deoxy-guanosine, 8-OH-guanine, and 8-NO2-cGMP) were detected in plasma samples following the one-month intervention. No significant modulation of DNA and lipid damage markers was documented among groups, apart from an effect of time for DNA strand breaks and some markers of lipid peroxidation. In conclusion, the consumption of coffee and cocoa-based confectionery containing coffee was apparently not able to affect oxidative stress markers. More studies are encouraged to better explain the findings obtained and to understand the impact of different dosages of these products on specific target groups.

Effects of phosphodiesterase V inhibition alone and in combination with BNP on cardiovascular and renal response to volume load in human preclinical diastolic dysfunction.

Preclinical diastolic dysfunction (PDD) results in impaired cardiorenal response to volume load (VL) which may contribute to the progression to clinical heart failure with preserved ejection fraction (HFpEF). The objective was to evaluate if phosphodiesterase V inhibition (PDEVI) alone or combination PDEVI plus B-type natriuretic peptide (BNP) administration will correct the impaired cardiorenal response to VL in PDD. A randomized double-blinded placebo-controlled cross-over study was conducted in 20 subjects with PDD, defined as left ventricular ejection fraction (LVEF) >50% with moderate or severe diastolic dysfunction by Doppler echocardiography and without HF diagnosis or symptoms. Effects of PDEVI with oral tadalafil alone and tadalafil plus subcutaneous (SC) BNP, administered prior to acute volume loading, were assessed. Tadalafil alone did not result in improvement in cardiac response to VL, as measured by LVEF, LV end diastolic volume, left atrial volume (LAV), or right ventricular systolic pressure (RVSP). Tadalafil plus SC BNP resulted in improved cardiac response to VL, with increased LVEF (4.1 vs. 1.8%, p = 0.08) and heart rate (4.3 vs. 1.6 bpm, p = 0.08), and reductions in both LAV (-4.3 ± 10.4 vs. 2.8 ± 6.6 ml, p = 0.03) and RVSP (-4.0 ± 3.0 vs. 2.1 ± 6.0 mmHg, p < 0.01) versus tadalafil alone. Plasma and urinary cyclic guanosine monophosphate (cGMP) excretion levels were higher (11.3 ± 12.3 vs. 1.7 ± 3.8 pmol/ml, 1851.0 ± 1386.4 vs. 173.4 ± 517.9 pmol/min, p < 0.01) with tadalafil plus SC BNP versus tadalafil alone. There was no improvement in renal response as measured by GFR, renal plasma flow, sodium excretion, and urine flow with tadalafil plus SC BNP compared to tadalafil alone. In subjects with PDD, tadalafil alone resulted in no improvement in cardiac adaptation, while tadalafil and SC BNP resulted in enhanced cardiac adaptation to VL. TRIAL REGISTRATION: ClinicalTrials.gov NCT01544998.

Aortic disease in Marfan syndrome is caused by overactivation of sGC-PRKG signaling by NO.

Thoracic aortic aneurysm, as occurs in Marfan syndrome, is generally asymptomatic until dissection or rupture, requiring surgical intervention as the only available treatment. Here, we show that nitric oxide (NO) signaling dysregulates actin cytoskeleton dynamics in Marfan Syndrome smooth muscle cells and that NO-donors induce Marfan-like aortopathy in wild-type mice, indicating that a marked increase in NO suffices to induce aortopathy. Levels of nitrated proteins are higher in plasma from Marfan patients and mice and in aortic tissue from Marfan mice than in control samples, indicating elevated circulating and tissue NO. Soluble guanylate cyclase and cGMP-dependent protein kinase are both activated in Marfan patients and mice and in wild-type mice treated with NO-donors, as shown by increased plasma cGMP and pVASP-S239 staining in aortic tissue. Marfan aortopathy in mice is reverted by pharmacological inhibition of soluble guanylate cyclase and cGMP-dependent protein kinase and lentiviral-mediated Prkg1 silencing. These findings identify potential biomarkers for monitoring Marfan Syndrome in patients and urge evaluation of cGMP-dependent protein kinase and soluble guanylate cyclase as therapeutic targets.

Local Action of Neprilysin Exacerbates Pressure Overload Induced Cardiac Remodeling.

[Figure: see text].

Sacubitril/Valsartan Improves Diastolic Function But Not Skeletal Muscle Function in a Rat Model of HFpEF.

The angiotensin receptor/neprilysin inhibitor Sacubitril/Valsartan (Sac/Val) has been shown to be beneficial in patients suffering from heart failure with reduced ejection fraction (HFrEF). However, the impact of Sac/Val in patients presenting with heart failure with preserved ejection fraction (HFpEF) is not yet clearly resolved. The present study aimed to reveal the influence of the drug on the functionality of the myocardium, the skeletal muscle, and the vasculature in a rat model of HFpEF. Female obese ZSF-1 rats received Sac/Val as a daily oral gavage for 12 weeks. Left ventricle (LV) function was assessed every four weeks using echocardiography. Prior to organ removal, invasive hemodynamic measurements were performed in both ventricles. Vascular function of the carotid artery and skeletal muscle function were monitored. Sac/Val treatment reduced E/é ratios, left ventricular end diastolic pressure (LVEDP) and myocardial stiffness as well as myocardial fibrosis and heart weight compared to the obese control group. Sac/Val slightly improved endothelial function in the carotid artery but had no impact on skeletal muscle function. Our results demonstrate striking effects of Sac/Val on the myocardial structure and function in a rat model of HFpEF. While vasodilation was slightly improved, functionality of the skeletal muscle remained unaffected.

Predialysis and Dialysis Therapies Differently Affect Nitric Oxide Synthetic Pathway in Red Blood Cells from Uremic Patients: Focus on Peritoneal Dialysis.

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD; hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.

Cyclic guanosine monophosphate and 10-year change in left ventricular mass: the Multi-Ethnic Study of Atherosclerosis (MESA).

Purpose: Cyclic guanosine monophosphate (cGMP) is a second messenger for natriuretic peptide (NP) and nitric oxide pathways; its enhancement a target for heart failure and cardiovascular disease (CVD). We evaluated whether plasma cGMP was associated with change in left ventricular mass (LVM) among individuals free of CVD and if this differed by sex.Methods and Results: In 611 men and 612 women aged 45-84 years with plasma cGMP measured at baseline and cardiac MRI performed at baseline and 10 years later, we tested associations of cGMP [log-transformed, per 1 SD increment] with LVM, adjusting for CVD risk factors and N-terminal pro-B-type-NP (NT-proBNP). Participants had mean (SD) age of 63.1(8.5) years and cGMP 4.8(2.6) pmol/mL. Cross-sectionally, higher cGMP was associated with lesser LVM, non-lin- early. In contrast, longitudinally, higher cGMP was associated with increase in LVM [1.70g (0.61, 2.78)] over 10 years. Higher cGMP was associated with greater LVM change in men [2.68g (1.57, 3.79)] but not women [0.24g ((-0.92, 1.39); p-interaction < 0.001].Conclusion: In conclusion, in a community-based cohort, higher cGMP levels were associated with increase in LVM over 10 years independent of CVD risk factors and NT-proBNP in men, perhaps reflecting compensatory changes. Further studies are needed to understand mechanistic roles of cGMP in LV remodelling and associated sex differences.

CRD-733, a Novel PDE9 (Phosphodiesterase 9) Inhibitor, Reverses Pressure Overload-Induced Heart Failure.

BACKGROUND: Augmentation of NP (natriuretic peptide) receptor and cyclic guanosine monophosphate (cGMP) signaling has emerged as a therapeutic strategy in heart failure (HF). cGMP-specific PDE9 (phosphodiesterase 9) inhibition increases cGMP signaling and attenuates stress-induced hypertrophic heart disease in preclinical studies. A novel cGMP-specific PDE9 inhibitor, CRD-733, is currently being advanced in human clinical studies. Here, we explore the effects of chronic PDE9 inhibition with CRD-733 in the mouse transverse aortic constriction pressure overload HF model. METHODS: Adult male C57BL/6J mice were subjected to transverse aortic constriction and developed significant left ventricular (LV) hypertrophy after 7 days (P<0.001). Mice then received daily treatment with CRD-733 (600 mg/kg per day; n=10) or vehicle (n=17), alongside sham-operated controls (n=10). RESULTS: CRD-733 treatment reversed existing LV hypertrophy compared with vehicle (P<0.001), significantly improved LV ejection fraction (P=0.009), and attenuated left atrial dilation (P<0.001), as assessed by serial echocardiography. CRD-733 prevented elevations in LV end diastolic pressures (P=0.037) compared with vehicle, while lung weights, a surrogate for pulmonary edema, were reduced to sham levels. Chronic CRD-733 treatment increased plasma cGMP levels compared with vehicle (P<0.001), alongside increased phosphorylation of Ser273 of cardiac myosin binding protein-C, a cGMP-dependent protein kinase I phosphorylation site. CONCLUSIONS: The PDE9 inhibitor, CRD-733, improves key hallmarks of HF including LV hypertrophy, LV dysfunction, left atrial dilation, and pulmonary edema after pressure overload in the mouse transverse aortic constriction HF model. Additionally, elevated plasma cGMP may be used as a biomarker of target engagement. These findings support future investigation into the therapeutic potential of CRD-733 in human HF.

Additive protective effects of sacubitril/valsartan and bosentan on vascular remodelling in experimental pulmonary hypertension.

AIMS: Although right ventricular (RV) function is an important determinant of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), there is no treatment targeting directly the RV. We evaluate the efficacy of sacubitril/valsartan (LCZ 696) as add-on therapy to bosentan in rats with severe pulmonary hypertension (PH). METHODS AND RESULTS: Combination therapy of LCZ 696 and bosentan has additive vascular protective effects against the pulmonary vascular remodelling and PH in two preclinical models of severe PH. Compared with monotherapy, co-treatment of LCZ 696 (30 or 68 mg/kg/day for 2 weeks, per os) and bosentan (100 mg/kg/day for 2 weeks, per os) started 7 days after monocrotaline (MCT) injection substantially reduces pulmonary pressures, vascular remodelling, and RV hypertrophy and fibrosis in rats. Consistent with these observations, co-treatment of rats with established PH induced by sugen/hypoxia (SuHx) with LCZ 696 (30 mg/kg/day for 3 weeks, per os) and bosentan (100 mg/kg/day for 3 weeks, per os) started 5 weeks after Sugen injection partially attenuate total pulmonary vascular resistance and cardiovascular structures. We also obtained evidence showing that LCZ 696 has anti-proliferative effect on cultured human pulmonary artery smooth muscle cells derived from patients with idiopathic PAH, an effect that is more pronounced in presence of bosentan. Finally, we found that the plasma levels of atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (cGMP) are higher in rats co-treated with LCZ 696 (30 mg/kg/day) and bosentan (100 mg/kg/day) than in MCT and SuHx rats treated with vehicle. CONCLUSION: Dual therapy with LCZ 696 plus bosentan proved significantly superior beneficial effect to LCZ 696 or bosentan alone on vascular remodelling and severity of experimental PH.

Natriuretic peptide analogues with distinct vasodilatory or renal activity: integrated effects in health and experimental heart failure.

AIMS: Management of acute decompensated heart failure (ADHF) requires disparate treatments depending on the state of systemic/peripheral perfusion and the presence/absence of expanded body-fluid volumes. There is an unmet need for therapeutics that differentially treat each aspect. Atrial natriuretic peptide (ANP) plays an important role in blood pressure and volume regulation. We investigate for the first time the integrated haemodynamic, endocrine and renal effects of human ANP analogues, modified for exclusive vasodilatory (ANP-DRD) or diuretic (ANP-DGD) activities, in normal health and experimental ADHF. METHODS AND RESULTS: We compared the effects of incremental infusions of ANP analogues ANP-DRD and ANP-DGD with native ANP, in normal (n = 8) and ADHF (n = 8) sheep. ANP-DRD administration increased plasma cyclic guanosine monophosphate (cGMP) in association with dose-dependent reductions in arterial pressure in normal and heart failure (HF) sheep similarly to ANP responses. In contrast to ANP, which in HF produced a diuresis/natriuresis, this analogue was without significant renal effect. Conversely, ANP-DGD induced marked stepwise increases in urinary cGMP, urine volume, and sodium excretion in HF comparable to ANP, but without accompanying vasodilatory effects. All peptides increased packed cell volume relative to control in both states, and in HF, decreased left atrial pressure. In response to ANP-DRD-induced blood pressure reductions, plasma renin activity rose compared to control only during the high dose in normals, and not at all in HF-suggesting relative renin inhibition, with no increase in aldosterone in either state, whereas renin and aldosterone were both significantly reduced by ANP-DGD in HF. CONCLUSION: These ANP analogues exhibit distinct vasodilatory (ANP-DRD) and diuretic/natriuretic (ANP-DGD) activities, and therefore have the potential to provide precision therapy for ADHF patients with differing pathophysiological derangement of pressure-volume homeostasis.

Safety, pharmacodynamic, and pharmacokinetic characterization of vericiguat: results from six phase I studies in healthy subjects.

PURPOSE: To characterize the safety, pharmacodynamics, and pharmacokinetics (PK) of vericiguat in healthy males. METHODS: Six phase I studies were conducted in European, Chinese, and Japanese males. Subjects received oral vericiguat as a single dose (0.5-15.0 mg solution [for first-in-human study] or 1.25-10.0 mg immediate release [IR tablets]) or multiple doses (1.25-10.0 mg IR tablets once daily [QD] or 5.0 mg IR tablets twice daily for 7 consecutive days). Bioavailability and food effects on vericiguat PK (IR tablets) were also studied in European subjects. RESULTS: Overall, 255 of 265 randomized subjects completed their respective studies. There were no deaths or serious adverse events. Vericiguat was generally well tolerated at doses ≤ 10.0 mg. In the first-in-human study, the most frequent drug-related adverse events were headache and postural dizziness (experienced by five subjects each [7.2%]). Three of four subjects who received vericiguat 15.0 mg (oral solution, fasted) experienced orthostatic reactions. Vericiguat (≤ 10.0 mg, IR tablets) was rapidly absorbed (median time to reach maximum plasma concentration ≤ 2.5 h [fasted]) with a mean half-life of about 22.0 h (range 17.9-27.0 h for single and multiple doses). No evidence for deviation from dose proportionality or unexpected accumulation was observed. Administration of vericiguat 5.0 mg IR tablets with food increased bioavailability by 19% (estimated ratio 119% [90% confidence interval]: 108; 131]), reduced PK variability, and prolonged vericiguat absorption relative to the fasted state. CONCLUSION: In general, vericiguat was well tolerated. These results supported further clinical evaluation of vericiguat QD in patients with heart failure. REGISTRY NUMBERS: EudraCT: 2011-001627-21; EudraCT: 2012-000953-30.

Adoptive transfer of placental ischemia-stimulated natural killer cells causes a preeclampsia-like phenotype in pregnant rats.

PROBLEM: The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of preeclampsia including maternal hypertension, intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). While we have previously shown a 5-fold higher cytotoxicity of RUPP NKs versus normal pregnant NKs, their role in RUPP pathophysiology remains unclear. In this study, we tested the hypotheses that (1) adoptive transfer of RUPP-stimulated NKs will induce maternal hypertension and IUGR in normal pregnant control (Sham) rats and (2) adoptive transfer of Sham NKs will attenuate maternal hypertension and IUGR in RUPP rats. METHOD OF STUDY: On gestation day (GD)14, vehicle or 5 × 106 RUPP NKs were infused i.v. into a subset of Sham rats (Sham+RUPP NK), and vehicle or 5 × 106 Sham NKs were infused i.v. into a subset of RUPP rats (RUPP+Sham NK; n = 12/group). On GD18, Uterine Artery Resistance Index (UARI) was measured. On GD19, mean arterial pressure (MAP) was measured, animals were sacrificed, and blood and tissues were collected for analysis. RESULTS: Adoptive transfer of RUPP NKs into Sham rats resulted in elevated NK activation, UARI, placental oxidative stress, and preproendothelin expression as well as reduced circulating nitrate/nitrite. This led to maternal hypertension and IUGR. RUPP recipients of Sham NKs demonstrated normalized NK activation, sFlt-1, circulating and placental VEGF, and UARI, which led to improved maternal blood pressure and normal fetal growth. CONCLUSION: These data suggest a direct role for cNKs in causing preeclampsia pathophysiology and a role for normal NKs to improve maternal outcomes and IUGR during late gestation.

NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis.

OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

[Influence of acupuncture combined with rehabilitation therapy on limb motor function and serum cAMP and cGMP in patients with hemiplegia of cerebral ischemic stroke].

OBJECTIVE: To evaluate the therapeutic effect of the combined treatment with balance acupuncture therapy and exercise re-learning rehabilitation therapy and the impact on serum cAMP and cGMP in the patients with hemiplegia of cerebral ischemic stroke. METHODS: A total of 90 patients of hemiplegia of cerebral ischemic stroke were randomized into an observation group and a control group, 45 cases in each one. All of the patients in the two groups received health education, diet guidance, routine symptomatic treatment as well as exercise re-learning rehabilitation therapy. Additionally, in the observation group, balance acupuncture therapy was applied, in which, the acupoints on the yang aspect of the human body, on the governor vessel and bladder meridian were adopted in the morning and those on the yin aspect of the human body, on the conception vessel and kidney meridian were stimulated in the afternoon. In the control group, the regular acupuncture was given. In the two groups, both acupuncture and rehabilitation therapies were given 5 days a week, 2 week-treatment as one course and totally 2 courses were required. Separately, before and after treatment, the score of Fugl-Meyer assessment (FMA) and the score of Chinese stroke scale (CSS) were recorded, the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) detected in serum and the clinical therapeutic effect were evaluated in the two groups. RESULTS: After treatment, FMA score was increased in the patients of either of the groups as compared with that before treatment (P<0.01) and CSS score decreased as compared with that before treatment (P<0.01). After treatment, FMA score in the observation group was higher than that in the control group (P<0.01) and CSS score was lower than the control group (P<0.01). After treatment, the level of serum cAMP of the patients in either of the groups was increased as compared with that before treatment (P<0.01) and that of cGMP decreased as compared with that before treatment (P<0.01). After treatment, the level of cAMP in the observation group was higher than that in the control group (P<0.01) and that of cGMP was lower than the control group (P<0.01). The total effective rate was 93.3% (42/45) in the observation group, better than 73.3% (33/45) in the control group (P<0.01). CONCLUSION: The balance acupuncture therapy combined with exercise re-learning rehabilitation effectively improves the motor function of the affected limb, relieves injury and regulate the levels of serum cAMP and cGMP in the patients with hemiplegia of ischemic stroke.

Heart is the Target Organ of Endogenous Cardiac Natriuretic Peptides.

This study aimed to evaluate whether the heart is the target organ of endogenous atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in patients with heart failure (HF) with reduced ejection fraction (HFrEF).We measured the plasma levels of cyclic guanosine monophosphate (cGMP), which is a second messenger of ANP and BNP, in the aortic root (AO) and coronary sinus (CS) in 237 patients with HFrEF. Plasma levels of cGMP were significantly higher in the CS than those in the AO in 237 patients with HFrEF (10.0 ± 4.5 versus 10.5 ± 4.3 pmoL/mL, P < 0.0001) and were significantly higher in the CS than those in the AO (8.0 ± 3.6 versus 8.9 ± 3.8 pmoL/mL, P < 0.0001) in mild HF patients (New York Heart Association (NYHA) II, n = 114), but there was no difference in plasma cGMP between the AO and the CS (11.9 ± 4.4 versus 11.9 ± 4.3 pmoL/mL, NS) in severe HF patients (NYHA III-IV, n = 123). In mild HF patients, log (ANP + BNP) in the AO was an independent predictor of (CS-AO) cGMP among hemodynamics and nitrate therapy. There was a significant correlation between log [(CS-AO) ANP + (CS-AO) BNP] and (CS-AO) cGMP (r = 0.455, P < 0.0001) in mild HF patients.These findings indicate that cGMP is produced from the failing heart and that the heart is the target organ of endogenous ANP and BNP in patients with HFrEF. In severe HF patients, cGMP production may be attenuated because of the downregulation of biological receptors and/or increased cGMP degradation in the failing heart.

Low expression of vascular endothelial growth factor and high serum level of cyclic guanine monophosphate as the risk factors of femoral head osteonecrosis in alcohol-exposed Wistar rat.

PURPOSE: Severe damage to the femoral head in patients with osteonecrosis has a high impact on morbidity. Despite early diagnosis, the treatment outcome is still unsatisfactory. This study aimed to explore the expression of vascular endothelial growth factor (VEGF) and cyclic guanine monophosphate (cGMP) serum level as the risk factors of femoral head osteonecrosis in alcohol-exposed Wistar rats. METHODS: This was an experimental study using randomized post-test only control group design, with samples using 10-14 weeks Wistar male rats. Rats were then divided into 6 groups: 3 groups without intervention, and 3 groups with intervention using 40% alcohol given perorally. Each one group from intervention and control group was euthanized by the end of the week for 3 consecutive weeks. Proximal femurs were examined under microscope for osteonecrosis, immunohistochemically for VEGF, and blood serum for cGMP levels. RESULTS: VEGF expression in the femoral head of alcohol-exposed Wistar rats was lower than those not exposed to alcohol (p < 0.005). Blood serum cGMP levels of alcohol-exposed Wistar rats were higher than those not exposed to alcohol (p < 0.005). The number of necrotic osteocytes in the femoral head of Wistar rats exposed to alcohol was greater than those not exposed to alcohol (p < 0.005). There are significant differences between VEGF, cGMP levels, and number of necrotic osteocytes in the control group and treatment at 1st, 2nd, and 3rd week (p < 0.005). CONCLUSIONS: Based on the result of this study, VEGF and cGMP may be considered as diagnostic biomarkers for alcohol-induced femoral head osteonecrosis.

Cyclic Guanosine Monophosphate and Risk of Incident Heart Failure and Other Cardiovascular Events: the ARIC Study.

Background Cyclic guanosine monophosphate (cGMP) is a second messenger regulated through natriuretic peptide and nitric oxide pathways. Stimulation of cGMP signaling is a potential therapeutic strategy for heart failure with preserved ejection fraction (HFpEF) and atherosclerotic cardiovascular disease (ASCVD). We hypothesized that plasma cGMP levels would be associated with lower risk for incident HFpEF, any HF, ASCVD, and coronary heart disease (CHD). Methods and Results We conducted a case-cohort analysis nested in the ARIC (Atherosclerosis Risk in Communities) study. Plasma cGMP was measured in 875 participants at visit 4 (1996-1998), with oversampling of incident HFpEF cases. We used Cox proportional hazard models to assess associations of cGMP with incident HFpEF, HF, ASCVD (CHD+stroke), and CHD. The mean (SD) age was 62.4 (5.6) years and median (interquartile interval) cGMP was 3.4 pmol/mL (2.4-4.6). During a median follow-up of 9.9 years, there were 283 incident cases of HFpEF, 329 any HF, 151 ASCVD, and 125 CHD. In models adjusted for CVD risk factors, the hazard ratios (95% CI) associated with the highest cGMP tertile compared with lowest for HFpEF, HF, ASCVD, and CHD were 1.88 (1.17-3.02), 2.18 (1.18-4.06), 2.84 (1.44-5.60), and 2.43 (1.19-5.00), respectively. In models further adjusted for N-terminal-proB-type natriuretic peptide, associations were attenuated for HFpEF and HF but remained statistically significant for ASCVD (2.56 [1.26-5.20]) and CHD (2.25 [1.07-4.71]). Conclusions Contrary to our hypothesis, higher cGMP levels were associated with incident CVD in a community-based cohort. The associations of cGMP with HF or HFpEF may be explained by N-terminal-proB-type natriuretic peptide, but not for ASCVD and CHD.

Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments.

The hypothesis of decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD) proposes that multiple factors leading to decreased NO production and increased consumption contributes to vaso-occlusion, pulmonary hypertension, and pain. The anion nitrite is central to NO physiology as it is an end product of NO metabolism and serves as a reservoir for NO formation. However, there is little data on nitrite levels in SCD patients and its relationship to pain phenotype. We measured nitrite in SCD subjects and examined its relationship to SCD pain. In SCD subjects, median whole blood, red blood cell and plasma nitrite levels were higher than in controls, and were not associated with pain burden. Similarly, Townes and BERK homozygous SCD mice had elevated blood nitrite. Additionally, in red blood cells and plasma from SCD subjects and in blood and kidney from Townes homozygous mice, levels of cyclic guanosine monophosphate (cGMP) were higher compared to controls. In vitro, hemoglobin concentration, rather than sickle hemoglobin, was responsible for nitrite metabolism rate. In vivo, inhibition of NO synthases and xanthine oxidoreductase decreased nitrite levels in homozygotes but not in control mice. Long-term nitrite treatment in SCD mice further elevated blood nitrite and cGMP, worsened anemia, decreased platelets, and did not change pain response. These data suggest that SCD in humans and animals is associated with increased nitrite/NO availability, which is unrelated to pain phenotype. These findings might explain why multiple clinical trials aimed at increasing NO availability in SCD patients failed to improve pain outcomes.

Associations Between the Cyclic Guanosine Monophosphate Pathway and Cardiovascular Risk Factors: MESA.

Background cGMP mediates numerous cardioprotective functions and is a potential therapeutic target for cardiovascular disease. Preclinical studies suggest that plasma cGMP is reflective of natriuretic peptide stimulation. Epidemiologic associations between cGMP and natriuretic peptide, as well as cardiovascular disease risk factors, are unknown. Methods and Results We measured plasma cGMP in 542 men and 496 women free of cardiovascular disease and heart failure in MESA (Multi-Ethnic Study of Atherosclerosis). Cross-sectional associations of N-terminal pro-B type natriuretic peptide, sex hormones, and cardiovascular disease/heart failure risk factors with log(cGMP) were analyzed using multivariable linear regression models. Mean (SD) cGMP was 4.7 (2.6) pmol/mL, with no difference between the sexes. After adjusting for cardiovascular risk factors, N-terminal pro-B type natriuretic peptide was significantly positively associated with cGMP (P<0.05). Higher blood pressure and lower estimated glomerular filtration rate were associated with higher cGMP (P<0.05). Triglyceride levels, total/high-density lipoprotein cholesterol ratio, presence of diabetes mellitus, and the homeostatic model assessment of insulin resistance were inversely associated with cGMP (P<0.05). Among women, free testosterone and dehydroepiandrosterone were inversely associated with cGMP, while sex hormone binding globulin was positively associated (P<0.05). Conclusions In a community-cohort, plasma cGMP was associated with natriuretic peptide signaling. Higher blood pressure and greater renal dysfunction were positively associated with cGMP, while adverse metabolic risk factors were inversely associated. Increased androgenicity in postmenopausal women was inversely associated with cGMP. These novel associations further our understanding of the role of cGMP in a general population.

Differential Regulation of ANP and BNP in Acute Decompensated Heart Failure: Deficiency of ANP.

OBJECTIVES: This study investigated the differential regulation of circulating atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in patients with acute decompensated heart failure (ADHF) and tested the hypothesis that a relative deficiency of ANP exists in a subgroup of patients with ADHF. BACKGROUND: The endocrine heart releases the cardiac hormones ANP and BNP, which play a key role in cardiovascular (CV), renal, and metabolic homeostasis. In heart failure (HF), both plasma ANP and BNP are increased as a compensatory homeostatic response to myocardial overload. METHODS: ANP and BNP concentrations were measured in a small group of patients with ADHF (n = 112). To support this study's goal, a total of 129 healthy subjects were prospectively recruited to establish contemporary normal values for ANP and BNP. Plasma 3',5'cyclic guanosine monophosphate (cGMP), ejection fraction (EF), and body mass index (BMI) were measured in these subjects. RESULTS: In cases of ADHF, 74% of patients showed elevated ANP and BNP. Importantly, 26% of patients were characterized as having normal ANP (21% of this subgroup had normal ANP and elevated BNP). Cyclic GMP was lowest in the ADHF group with normal levels of ANP (p < 0.001), whereas BMI and EF were inversely related to ANP levels (p = 0.003). CONCLUSIONS: Among a subgroup of patients hospitalized with ADHF, the presence of an ANP deficiency is consistent with a differential regulation of ANP and BNP and suggests the existence of a potentially compromised compensatory cardiac endocrine response. These findings have implications for the pathophysiology, diagnostics, and therapeutics of human HF.