Mitochondria-targeted antioxidant mitoquinone attenuates liver inflammation and fibrosis in cirrhotic rats.

In liver cirrhosis, oxidative stress plays a major role in promoting liver inflammation and fibrosis. Mitochondria dysregulation is responsible for excessive reactive oxygen species production. Therefore, in an experimental model of cirrhosis, we investigated the effect of mitochondria-targeted antioxidant mitoquinone. Liver cirrhosis was induced in Spraque-Dawley rats by common bile duct ligation (CBDL). Mitoquinone (10 mg·kg-1·day-1, oral gavage) or vehicle was administered from 3rd to 28th day after CBDL, when animals were euthanized; liver oxidative stress, inflammation, fibrosis, mitophagy were evaluated; and in vivo and ex vivo hemodynamic studies were performed. In cirrhotic rats, mitoquinone prevented liver inflammation, hepatocyte necrosis, and fibrosis at histological examination; decreased circulating TNF-α, gene expression of transforming growth factor-ß1, collagen type 1a1, TNF-α, IL-6, IL-1ß, tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-2, and MMP-13; and reduced hepatic oxidative stress, as shown by reduced oxidative carbonylation of the proteins, by modulating antioxidants catalase, Mn superoxide dismutase, and Cu/Zn superoxide dismutase. Furthermore, mitoquinone attenuated apoptosis by reducing hepatic protein expression of cleaved caspase-3. A selective removal of dysfunctional mitochondria was improved by mitoquinone, as shown by the increase in Parkin translocation to mitochondria. Treatment with mitoquinone normalized the weight of the spleen; however, it increased portal blood flow and reduced splenic artery intrahepatic resistance, suggesting an effect on resistance index. Mitochondria-targeted antioxidant mitoquinone improves liver inflammation and fibrosis in cirrhotic rats by reducing hepatic oxidative stress, preventing apoptosis, and promoting removal of dysfunctional mitochondria. Therefore, it may represent a promising strategy for the prevention and treatment of liver cirrhosis.

Incidence trends of vulvar squamous cell carcinoma in Italy from 1990 to 2015.

OBJECTIVE: The incidence of vulvar squamous cell carcinoma has increased for decades in most Western countries - a trend virtually restricted to women aged <50 or 60 years. In southern Europe, conversely, the trends have been insufficiently studied. This article reports a study from Italy. METHOD: Thirty-eight local cancer registries, currently covering 15,274,070 women, equivalent to 49.2% of the Italian national female population, participated. Invasive cancers registered between 1990 and 2015 with an International Classification of Diseases for Oncology, 3rd revision, topography code C51 and morphology codes compatible with vulvar squamous cell carcinoma (n = 6294) were eligible. Incidence trends were analysed using joinpoint regression models, with calculation of the estimated annual percent change (EAPC), and age-period-cohort models. RESULTS: Total incidence showed a regular and significant decreasing trend (EAPC, -0.96; 95% confidence interval (CI), -1.43 to -0.48). This was entirely accounted for by women aged ≥60 years (EAPC, -1.34; 95% CI, -1.86 to -0.81). For younger women, the EAPC between 1990 and 2012 was 1.20 (95% CI, 0.34 to 2.06) with a non-significant acceleration thereafter. This pattern did not vary substantially in a sensitivity analysis for the effect of geographic area and duration of the registry. The age-period-cohort analysis revealed a risk decrease in cohorts born between 1905 and 1940 and a new increase in cohorts born since 1945. CONCLUSIONS: The decreasing trend observed among older women and the resulting decrease in total rate are at variance with reports from most Western countries. Age-period-cohort analysis confirmed a decreasing trend for earliest birth cohorts and an opposite one for recent ones.

Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3.

INTRODUCTION AND OBJECTIVES: SerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics. MATERIAL AND METHODS: Different hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl4) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose-response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment. RESULTS: In SerpinB3 transgenic mice, cardiac output (51.6±21.5 vs 30.1±10.8ml/min, p=0.003), hepatic artery pulsatility index (0.85±0.13 vs 0.65±0.11, p<0.001) and portal vein blood flow (5.3±3.2 vs 3.1±1.8ml/min, p=0.03) were significantly increased, compared to controls. In vitro, recombinant SerpinB3 had no direct hemodynamic effect on mesenteric arteries, but it increased their sensitivity to phenylephrine-mediated vasoconstriction (p<0.01). This effect was suppressed by inhibiting angiotensin II type-1 receptors. CONCLUSIONS: In transgenic mice, SerpinB3 is associated with a hyperdynamic circulatory syndrome-like pattern, possibly mediated by angiotensin receptors.

Inhibition of epoxyeicosatrienoic acid production in rats with cirrhosis has beneficial effects on portal hypertension by reducing splanchnic vasodilation.

UNLABELLED: In cirrhosis, 11,12-epoxyeicosatrienoic acid (EET) induces mesenteric arterial vasodilation, which contributes to the onset of portal hypertension. We evaluated the hemodynamic effects of in vivo inhibition of EET production in experimental cirrhosis. Sixteen control rats and 16 rats with carbon tetrachloride-induced cirrhosis were studied. Eight controls and eight rats with cirrhosis were treated with the specific epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH; 20 mg/kg/day) for 3 consecutive days. Portal blood flow and renal and splenic resistive indexes were calculated through echographic measurements, while portal and systemic pressures were measured through polyethylene-50 catheters. Small resistance mesenteric arteries were connected to a pressure servo controller in a video-monitored perfusion system, and concentration-response curves to phenylephrine and acetylcholine were evaluated. EET levels were measured in tissue homogenates of rat liver, kidney, and aorta, using an enzyme-linked immunosorbent assay. Urinary Na(+) excretion function was also evaluated. In rats with cirrhosis, treatment with MS-PPOH significantly reduced portal blood flow and portal pressure compared to vehicle (13.6 ± 5.7 versus 25.3 ± 7.1 mL/min/100 g body weight, P < 0.05; 9.6 ± 1.1 versus 12.2 ± 2.3 mm Hg, P < 0.05; respectively) without effects on systemic pressure. An increased response to acetylcholine of mesenteric arteries from rats with cirrhosis (50% effect concentration -7.083 ± 0.197 versus -6.517 ± 0.73 in control rats, P < 0.05) was reversed after inhibition of EET production (-6.388 ± 0.263, P < 0.05). In liver, kidney, and aorta from animals with cirrhosis, treatment with MS-PPOH reversed the increase in EET levels. In both controls and rats with cirrhosis, MS-PPOH increased urinary Na(+) excretion. CONCLUSION: In rats with cirrhosis, in vivo inhibition of EET production normalizes the response of mesenteric arteries to vasodilators, with beneficial effects on portal hypertension. (Hepatology 2016;64:923-930).

EETs and HO-1 cross-talk.

Epoxygenase-dependent metabolites of arachidonc acid, EETs and the heme-oxygenase (HO)-1/carbon monoxide/bilverdin system share similarities in their activity and mediators. They control endothelial function, dilating small arterial vessels, decrease blood pressure, protect the heart from ischemic and hypertensive cardiopathy, control renal circulation and function, promote angiogenesis and organ regeneration, oppose oxidative stress and inflammation, improve diabetes and obesity, have protective effects on the liver, and participate in portal hypertension. Furthermore, EETs induce HO-1, and inhibition of HO-1 abolishes most of the effects of EETs. Thus, a close interaction between the two systems exists, and is relevant in view of their therapeutic potential.

Arachidonic acid metabolites and endothelial dysfunction of portal hypertension.

Increased resistance to portal flow and increased portal inflow due to mesenteric vasodilatation represent the main factors causing portal hypertension in cirrhosis. Endothelial cell dysfunction, defined as an imbalance between the synthesis, release, and effect of endothelial mediators of vascular tone, inflammation, thrombosis, and angiogenesis, plays a major role in the increase of resistance in portal circulation, in the decrease in the mesenteric one, in the development of collateral circulation. Reduced response to vasodilators in liver sinusoids and increased response in the mesenteric arterioles, and, viceversa, increased response to vasoconstrictors in the portal-sinusoidal circulation and decreased response in the mesenteric arterioles are also relevant to the pathophysiology of portal hypertension. Arachidonic acid (AA) metabolites through the three pathways, cyclooxygenase (COX), lipoxygenase, and cytochrome P450 monooxygenase and epoxygenase, are involved in endothelial dysfunction of portal hypertension. Increased thromboxane-A2 production by liver sinusoidal endothelial cells (LSECs) via increased COX-1 activity/expression, increased leukotriens, increased epoxyeicosatrienoic acids (EETs) (dilators of the peripheral arterial circulation, but vasoconstrictors of the portal-sinusoidal circulation), represent a major component in the increased portal resistance, in the decreased portal response to vasodilators and in the hyper-response to vasoconstrictors. Increased prostacyclin (PGI2) via COX-1 and COX-2 overexpression, and increased EETs/heme-oxygenase-1/K channels/gap junctions (endothelial derived hyperpolarizing factor system) play a major role in mesenteric vasodilatation, hyporeactivity to vasoconstrictors, and hyper-response to vasodilators. EETs, mediators of liver regeneration after hepatectomy and of angiogenesis, may play a role in the development of regenerative nodules and collateral circulation, through stimulation of vascular endothelial growth factor (VEGF) inside the liver and in the portal circulation. Pharmacological manipulation of AA metabolites may be beneficial for cirrhotic portal hypertension.

Changes in gene expression of cytochrome P-450 in liver, kidney and aorta of cirrhotic rats.

INTRODUCTION: Liver cirrhosis is characterized by structural and hemodynamic changes that affect mainly the liver, the kidney and the vascular system. Cytochrome P-450 (CYP) is a variegated family of enzymes that, among many other activities, metabolize arachidonic acid to the vasoactive epoxyeicosatrienoic acids (EETs). AIM: To investigate in an animal model of cirrhosis the m-RNA expression of CYPs in liver, kidney and aorta and to evaluate the effect of epoxygenase inhibition by N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH). METHODS: In aorta, liver and kidney from 3 control, 3 cirrhotic and 6 cirrhotic rats treated with MS-PPOH, quantitative real-time PCR reactions were performed and the m-RNA expression of CYP2J3, CYP2J4, CYP2J10, CYP2C11, CYP2C12 and CYP2C23 was calculated. RESULTS: In cirrhotic rats, the gene expression of hepatic CYP2C11 and CYP2J10 was increased, of aortic CYP2J4 was increased, of aortic CYP2C12 was reduced and of renal CYP2C11 was increased. In cirrhotic rats, MS-PPOH reduced CYP2J10 hepatic and CYP2C11 renal gene expression to levels similar to the ones of control rats. CONCLUSIONS: Changes in CYPs gene expression may contribute to the hemodynamic alterations typical of cirrhosis. The altered gene expression of CYPs can, in some cases, be reversed by epoxygenase inhibition.

Trends in Net Survival from Vulvar Squamous Cell Carcinoma in Italy (1990-2015).

(1) Objective: In many Western countries, survival from vulvar squamous cell carcinoma (VSCC) has been stagnating for decades or has increased insufficiently from a clinical perspective. In Italy, previous studies on cancer survival have not taken vulvar cancer into consideration or have pooled patients with vulvar and vaginal cancer. To bridge this knowledge gap, we report the trend in survival from vulvar cancer between 1990 and 2015. (2) Methods: Thirty-eight local cancer registries covering 49% of the national female population contributed the records of 6274 patients. Study endpoints included 1- and 2-year net survival (NS) calculated using the Pohar-Perme estimator and 5-year NS conditional on having survived two years (5|2-year CNS). The significance of survival trends was assessed with the Wald test on the coefficient of the period of diagnosis, entered as a continuous regressor in a Poisson regression model. (3) Results: The median patient age was stable at 76 years. One-year NS decreased from 83.9% in 1990-2001 to 81.9% in 2009-2015 and 2-year NS from 72.2% to 70.5%. Five|2-year CNS increased from 85.7% to 86.7%. These trends were not significant. In the age stratum 70-79 years, a weakly significant decrease in 2-year NS from 71.4% to 65.7% occurred. Multivariate analysis adjusting for age group at diagnosis and geographic area showed an excess risk of death at 5|2-years, of borderline significance, in 2003-2015 versus 1990-2002. (4) Conclusions: One- and 2-year NS and 5|2-year CNS showed no improvements. Current strategies for VSCC control need to be revised both in Italy and at the global level.

Increased EETs participate in peripheral endothelial dysfunction of cirrhosis.

The hyperdynamic circulation of cirrhosis participates in the pathophysiology of portal hypertension. P450-dependent epoxyeicosatrienoic acids (EET) are potent vasodilators. We evaluated plasma levels of EETs in cirrhotic patients and the effect of epoxygenase and nitric oxide synthase (NOS) inhibition on skin blood flow, measured by laser Doppler flowmetry, in normal subjects and cirrhotic patients with and without ascites. Free plasma EETs were increased in cirrhotic patients compared to normal subjects, while the ratio between 8,9-, 11,12-, and 14-15-EET was the same. In cirrhotic patients without ascites, skin blood flow was significantly increased compared to normal subjects. In patients with ascites skin blood flow was significantly reduced compared to control subjects and patients without ascites. Inhibition of epoxygenase with miconazole and of NOS with L-NG-Nitroarginine methyl ester (L-NAME) decreased basal skin flow in normal subjects and in cirrhotic patients, the effect being higher in cirrhotic patients. Miconazole caused a further decrease in flow when administered with L-NAME, both in normal subjects and in cirrhotic patients. In conclusion, EETs participate in the control of peripheral circulation of normal subjects and in the pathophysiology of peripheral vasodilatation of cirrhotic patients with ascites.

Myocardial overexpression of ANKRD1 causes sinus venosus defects and progressive diastolic dysfunction.

AIMS: Increased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium. METHODS AND RESULTS: Ankrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are progressively impaired from embryonic to adult life. Postnatal transgenic myofibrils also present characteristic functional alterations: impaired compliance at neonatal stage and impaired lusitropism in adult hearts. Altogether, our combined analyses suggest that impaired embryonic remodelling and adult heart dysfunction in ANKRD1 transgenic mice present a common ground of initial cardiomyocyte defects, which are exacerbated postnatally. Molecular analysis showed transient activation of GATA4-Nkx2.5 transcription in early transgenic embryos and subsequent dynamic transcriptional modulation within titin gene. CONCLUSIONS: ANKRD1 is a fine mediator of cardiomyocyte response to haemodynamic load in the developing and adult heart. Increased ANKRD1 levels are sufficient to initiate an altered cellular phenotype, which is progressively exacerbated into a pathological organ response by the high ventricular workload during postnatal life. Our study defines for the first time a unifying picture for ANKRD1 role in heart development and disease and provides the first mechanistic link between ANKRD1 overexpression and cardiac disease onset.

Allogenic tissue-specific decellularized scaffolds promote long-term muscle innervation and functional recovery in a surgical diaphragmatic hernia model.

Congenital diaphragmatic hernia (CDH) is a neonatal defect in which the diaphragm muscle does not develop properly, thereby raising abdominal organs into the thoracic cavity and impeding lung development and function. Large diaphragmatic defects require correction with prosthetic patches to close the malformation. This treatment leads to a consequent generation of unwelcomed mechanical stress in the repaired diaphragm and hernia recurrences, thereby resulting in high morbidity and significant mortality rates. We proposed a specific diaphragm-derived extracellular matrix (ECM) as a scaffold for the treatment of CDH. To address this strategy, we developed a new surgical CDH mouse model to test the ability of our tissue-specific patch to regenerate damaged diaphragms. Implantation of decellularized diaphragmatic ECM-derived patches demonstrated absence of rejection or hernia recurrence, in contrast to the performance of a commercially available synthetic material. Diaphragm-derived ECM was able to promote the generation of new blood vessels, boost long-term muscle regeneration, and recover host diaphragmatic function. In addition, using a GFP + Schwann cell mouse model, we identified re-innervation of implanted patches. These results demonstrated for the first time that implantation of a tissue-specific biologic scaffold is able to promote a regenerating diaphragm muscle and overcome issues commonly related to the standard use of prosthetic materials. STATEMENT OF SIGNIFICANCE: Large diaphragmatic hernia in paediatric patients require application of artificial patches to close the congenital defect. The use of a muscle-specific decellularized scaffold in substitution of currently used synthetic materials allows new blood vessel growth and nerve regeneration inside the patch, supporting new muscle tissue formation. Furthermore, the presence of a tissue-specific scaffold guaranteed long-term muscle regeneration, improving diaphragm performance to almost complete functional recovery. We believe that diaphragm-derived scaffold will be key player in future pre-clinical studies on large animal models.

Heterotopic Implantation of Decellularized Pulmonary Artery Homografts In A Rodent Model: Technique Description and Preliminary Report.

PURPOSE: Despite a substantial amount of literature on tissue-guided regeneration, decellularization process, repopulation time points and stem cell turnover, more in-depth study on the argument is required. Currently, there are plenty of reports involving large animals, as well as clinical studies facing cardiac repair with decellularized homografts, but no exhaustive rodent models are described. The purpose of this study was to develop such a model in rats; preliminary results are also herein reported. MATERIAL AND METHODS: Fresh or decellularized pulmonary homografts from wild type rats were implanted in the abdominal aorta of green fluorescent protein positive rats. Three experimental groups were build up: sham, fresh homograft recipients and decellularized homograft recipients. The homograft decellularization process was performed with three cycles of detergent-enzymatic treatment protocol. Surgical technique of pulmonary homograft implantation and postoperative ultrasonographic evaluation were also reported; gross, histology and immunohistochemistry analysis on unimplanted and postoperative homografts were also carried out. RESULTS: The median total recipient operating time was 148 minutes, with a surgical success rate of 82%. The decellularization protocol resulted effective and showed a complete decellularization with intact extracellular matrix. At 15 days from surgery, the implanted decellularized pulmonary homografts exhibited cell repopulation in the outer media wall and partial endothelial lining in absence of rejection. CONCLUSIONS: Our technique is a feasible and reproducible model that can be fundamental for building a valid study for further exploitation on the field. Even in a short-term follow up, the decellularized pulmonary homografts showed autologous repopulation in absence of rejection.

The OPA1-dependent mitochondrial cristae remodeling pathway controls atrophic, apoptotic, and ischemic tissue damage.

Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy.

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

HO-1 induction improves the type-1 cardiorenal syndrome in mice with impaired angiotensin II-induced lymphocyte activation.

Type-1 cardiorenal syndrome, characterized by acute kidney dysfunction secondary to cardiac failure and renal arteriolar vasoconstriction, is mediated by the renin-angiotensin-aldosterone axis and sympathetic nervous system activation. Previous reports indicate that angiotensin II modulates immune function and causes recruitment and activation of T-lymphocytes. The goal of this study was to evaluate the effects of postischemic heart failure on renal morphology and circulation and the beneficial effects of heme oxygenase-1 (HO-1) induction in T-lymphocyte-suppressed severe combined immune deficiency (SCID) mice. Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with an HO-1 inducer, cobalt protoporphyrin, and with or without stannous mesoporphyrin, an inhibitor of HO activity. Heart and kidney function were studied 30 days after surgery. Fractional area change was reduced 30 days after surgery in both the C57 and SCID MI-groups as compared with their respective controls (P<0.01). Renal Pulsatility Index and renal injury were increased in C57 and SCID MI-groups compared with the sham group. HO-1 induction improved renal vasoconstriction as well as ameliorated renal injury in both the SCID and C57 MI-groups (P<0.01). However, improvement was more evident in SCID mice. In addition, our results showed that plasma creatinine, angiotensin II, and renin were significantly increased in the C57 and SCID MI-groups as compared with their respective controls. HO-1 induction decreased these parameters in both MI groups. Stannous mesoporphyrin reversed the beneficial effect of cobalt protoporphyrin in both mouse strains. The study demonstrates that T-lymphocyte suppression facilitated the HO-1-dependent improvement in the attenuation of type-1 cardiorenal syndrome.

Role of HO/CO in the Control of Peripheral Circulation in Humans.

Experimental studies show that the heme oxygenase/carbon monoxide system (HO/CO) plays an important role in the homeostasis of circulation and in the pathophysiology of hypertension. No data are available on its role in the control of peripheral circulation in humans. We evaluated the effects of inhibition of HO with stannous mesoporphyrin IX (SnMP) (200 µM) locally administered by iontophoresis, on human skin blood flow, evaluated by laser-Doppler flowmetry, in the presence and absence of nitric oxide synthase (NOS) inhibition with L-NG-Nitroarginine methyl ester (L-NAME) (100 µM). We also evaluated the effect of HO inhibition on vasodilatation induced by acetylcholine (ACh) and vasoconstriction caused by noradrenaline (NA). SnMP and L-NAME caused a similar 20-25% decrease in skin flow. After nitric oxide (NO) inhibition with L-NAME, HO inhibition with SnMP caused a further 20% decrease in skin perfusion. SnMP decreased vasodilatation induced by ACh by about 70%, while it did not affect vasoconstriction to NA. In conclusion, HO/CO participates in the control of peripheral circulation, independently from NO, and is involved in vasodilatation to ACh.

Splenic Doppler impedance indices estimate splenic congestion in patients with right-sided or congestive heart failure.

Splenic Doppler impedance indices are measurements of splenic congestion in chronic liver disease. It is not known whether they can also assess splenic congestion in patients affected by right-sided or congestive heart failure. We analyzed splanchnic hemodynamics with Doppler ultrasound and systemic hemodynamics with right-sided heart catheterization in patients with heart failure. Splenic pulsatility index (PI) was higher in patients with heart failure (48 patients) compared with healthy subjects (39 patients) (1.19 ± 0.41 vs. 0.73 ± 0.11, p < 0.0001) and was related to hepatic vein diameter (p = 0.02). Splenic PI was not related to systemic arterial pressure, cardiac output, systemic vascular resistance or splenic arterial resistance, whereas it was related to right atrial mean pressure (p = 0.0003) and to right ventricle end-diastolic pressure (p = 0.011) (34 patients). In conclusion, splenic PI is a measurement of splenic congestion caused by an increase in venous outflow resistance. It can estimate splenic congestion in patients with right-sided or congestive heart failure.