Paola Pesce

The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic, and Ischemic Tissue Damage

Summary 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.

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.

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.

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.

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

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)

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.

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.

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.

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.