Circulating levels of tumour necrosis factor-alpha and its soluble receptors are increased in the blood of patients with amyotrophic lateral sclerosis.

An immunologic pathogenesis for amyotrophic lateral sclerosis (ALS) has been recently proposed. We tested the whole tumour necrosis factor (TNF) system in the serum of 51 ALS patients at different stages of the disease and 36 healthy controls. Antigenic TNF-alpha and its soluble receptors (sTNF-Rs), measured by ELISA, were significantly higher in ALS patients than in healthy controls. However, biologically active TNF-alpha, corresponding to the sTNF-Rs-unbound trimeric TNFalpha molecule and assayed by its cytotoxic activity on a sensitive cell line, was similar between ALS patients and healthy controls. Neither antigenic TNF-alpha, bioactive TNF-alpha nor sTNF-Rs correlated with disease severity, disease duration, or weight loss. In conclusion, we reported an activation of the TNF system in ALS. The role of this activation in the pathogenesis of the disease remains elusive.

Serum from patients with severe heart failure downregulates eNOS and is proapoptotic: role of tumor necrosis factor-alpha.

BACKGROUND: Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis. METHODS AND RESULTS: We studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001). CONCLUSIONS: Serum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.

Induction of functional inducible nitric oxide synthase in monocytes of patients with congestive heart failure. Link with tumour necrosis factor-alpha.

AIMS: We studied the induction of monocytic inducible nitric oxide synthase expression and the tumour necrosis factor-alpha system in patients with congestive heart failure. METHODS AND RESULTS: Forty-three congestive heart failure patients and 15 healthy subjects were studied. Antigenic tumour necrosis factor-alpha and its soluble receptors, measured by ELISA, were increased in chronic heart failure and the increase was related to the clinical severity of the syndrome (tumour necrosis factor-alpha from 8.2+/-5.2 in NYHA class II to 18.2+/-7.2 in class III and 26.9+/-13.2 pg. ml(-1)in class IV, P<0.0001 classes III and IV vs class II; soluble tumour necrosis factor receptor I from 1.0+/-0.2 in class II to 2.3+/-1.1 in class III and 5.5+/-3.2 ng. ml(-1)in class IV, P<0.0001 classes III and IV vs class II; soluble tumour necrosis factor receptor II from 2.7+/-0.7 in class II to 4.9+/-1.9 in class III and 8.4+/-5.0 ng. ml(-1)in class IV, P<0.002 classes III and IV vs class II). Monocytic inducible nitric oxide synthase assessed by Western blot, was expressed only in congestive heart failure patients (13 out of 43). The association among monocytic inducible nitric oxide synthase expression, tumour necrosis factor-alpha system activation, neurohormones and other clinical parameters was studied. The univariate logistic regression showed that inducible nitric oxide synthase expression was strictly associated with NYHA class (P<0.05), antigenic tumour necrosis factor-alpha (P<0.01) and its soluble receptors (P<0.05). The multivariate analysis showed that antigenic tumour necrosis factor-alpha was the only predictor for monocytic inducible nitric oxide synthase expression (P<0.05, RR=2.75, CI 1. 34-5.43). CONCLUSIONS: Inducible nitric oxide synthase is expressed in circulating monocytes of patients with severe congestive heart failure. This phenomenon is linked to the activation of the tumour necrosis factor-alpha system.

Effects of chronic noradrenaline on the nitric oxide pathway in human endothelial cells.

Altered endothelium-dependent vasodilation has been observed in congestive heart failure (CHF), a disease characterized by a sustained adrenergic activation. The purpose of our study was to test the hypothesis that chronically elevated catecholamines influence the nitric oxide (NO) pathway in the human endothelium. Human umbilical vein endothelial cells (HUVEC) were exposed for 7 days to a concentration of noradrenaline (NA, 1 ng/mL) similar to that found in the blood of patients with CHF. Kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activity, measured by [3H]L-arginine to [3H]L-citrulline conversion, and protein expression of ecNOS and iNOS, assessed by Western blot analysis, were unaffected by chronic NA treatment. Furthermore, no changes in subcellular fraction-associated ecNOS were found; this indirectly shows that chronic NA did not cause phosphorylation of the enzyme. Moreover, [3H]L-arginine transport through the plasma membrane was conserved in chronically NA-treated cells. The data demonstrate that prolonged in vitro exposure to pathologic CHF-like NA does not affect the L-arginine: NO pathway in human endothelial cells.

Endothelial function and dysfunction in heart failure.

The endothelium controls vascular smooth muscle tone by secreting substances that cause relaxation and contraction. Under physiological, basal conditions the endothelium constantly releases nitric oxide, a process closely regulated by the effect of shear stress on endothelial cells. Recent data raise the possibility that, among many other substances, bradykinin also plays an important role in the regulation of vascular tone. Bradykinin is a vasodilator that increases the activity of constitutive nitric oxide. Congestive heart failure (CHF) is a complex clinical syndrome in which abnormal vascular endothelial function has been shown to occur at both the experimental and clinical level. The reduced nitric oxide-mediated vasodilation in CHF is multifactorial. Constitutive nitric oxide synthase is downregulated as shear stress is reduced. Vascular angiotensin-converting enzyme (ACE), which produces angiotensin II and inactivates bradykinin, is up-regulated. Angiotensin II is a powerful vasoconstrictor, and the reduced availability of bradykinin will further down-regulate constitutive nitric oxide synthase. The CHF-induced activation of tumour necrosis factor also leads to a down-regulation of constitutive nitric oxide synthase and to an increased rate of endothelial-cell apoptosis. These observations suggest that abnormalities of endothelial function in CHF may contribute to increased peripheral vasomotor tone both at rest and during exercise, and raise the possibility that the beneficial effects of ACE inhibition in CHF may be due in part to improved endothelial function.

Oxidative stress during myocardial ischaemia and heart failure.

Oxidative stress is a condition in which oxidant metabolites exert toxic effects because of their increased production or an altered cellular mechanism of protection. The heart needs oxygen but it is also susceptible to oxidative stress, which occurs during post-ischaemic reperfusion, for example. Ischaemia causes alterations in the defence mechanisms against oxygen free radicals. At the same time, production of oxygen free radicals increases. In man, there is evidence of oxidative stress during surgical reperfusion of the whole heart, or after thrombolysis, and it is related to transient left ventricular dysfunction or stunning. At present, there are few data on oxidative stress in the failing heart. It is not clear whether the defence mechanisms of the myocyte are altered or whether the production of oxygen free radicals is increased, or both. Recent data have shown a close link between oxidative stress and apoptosis. Importantly, tumour necrosis factor causes a rapid rise in intracellular reactive oxygen intermediates and apoptosis. This series of events is not confined to the myocytes, but also occurs at the level of endothelium, where tumour necrosis factor causes expression of inducible nitric oxide synthase, production of the reactive radical nitric oxide, oxidative stress and apoptosis. The immunological response to heart failure may result in endothelial and myocyte dysfunction through oxidative stress-mediated apoptosis. A better understanding of these mechanisms may lead to novel therapeutic strategies.

Aorta and skeletal muscle NO synthase expression in experimental heart failure.

Nitric oxide (NO), the free radical that accounts for the biological activity of endothelium-derived relaxing factor, is synthesized from L-arginine by NO synthase (NOS). There is evidence that NO availability is reduced in the peripheral vasculature of patients with congestive heart failure (CHF). The aim of this study was to investigate the expression of NOS in the descending aorta and in the skeletal muscles of rats subjected to heart failure. The alkaloid, monocrotaline, was used to induce pulmonary hypertension and cardiac failure in rats. The expression of both the constitutive (ecNOS) and the inducible (iNOS) isoforms of the enzyme was assessed by Western blot analysis. In CHF animals, the ecNOS location in the aorta is altered: the endothelial protein expression is substantially reduced (from 0.083 +/- 0.012 to 0.003 +/- 0.004 OD/microgram total proteins, P < 0.001) whereas the expression of ecNOS in the smooth muscle is increased (from 0.024 +/- 0.004 to 0.059 +/- 0.009 OD/ microgram total proteins, P < 0.01). The total aortic ecNOS is diminished in CHF respect to control animals (0.062 +/- 0.009 v 0.107 +/- 0.013 OD/microgram total proteins, P < 0.01). On the contrary, no difference in ecNOS protein expression was observed in the extensor digitorum longus and soleus muscles. Furthermore, iNOS was not detected in any of the tissues considered. In conclusion, experimental CHF causes a re-setting of the ecNOS protein expression in the descending aorta but not in skeletal muscles. The reduced abundance of ecNOS in the aortic endothelium is consistent with the impairment of the vasodilating function reported in patients with CHF.

[Activation and role of the tumor necrosis factor-alpha in congestive heart failure]. / Attivazione e ruolo del fattore di necrosi tumorale alfa nello scompenso cardiaco congestizio.

Recently, an activation of the immune system has been demonstrated in congestive heart failure (CHF). Aim of this study was to evaluate the effects of CHF on the activation of alpha tumor necrosis factor (TNF-alpha), a pleiotropic cytokine. Since the soluble forms of the TNF membrane receptors, sTNF-RI and sTNF-RII, have been shown to modulate TNF-alpha biological activity, we determined antigenic TNF-alpha, bioactive TNF-alpha, sTNF-RI and sTNF-RII in 52 patients with varying degrees of CHF (NYHA functional class II, III, IV). The etiology of CHF was coronary artery disease in 51% of the patients, idiopathic dilated cardiomyopathy in 38% and valvular disease in 11%. All patients were treated with ACE-inhibitors, digoxin and inotropic agents. Antigenic TNF-alpha was significantly increased in NYHA functional class IV patients (from 12.1 +/- 7.6 to 38.5 +/- 12.4 pg/ml, p < 0.001) whereas cytotoxic activity was always under the detection limit of the assay (100 pg/ml). Soluble TNF receptors were significantly elevated in NYHA functional class IV patients: sTNF-RI increased from 1.27 +/- 0.48 to 4.54 +/- 2.11 ng/ml (p < 0.001) and sTNF-RII from 2.25 +/- 0.55 to 7.78 +/- 2.13 ng/ml (p < 0.001). The possible modulation of TNF-alpha biological activity by the soluble receptors was investigated by means of spiking experiments after addition of 625 pg/ml human recombinant TNF-alpha to each serum sample. The biological activity of the added TNF-alpha was significantly inhibited by the high levels of soluble receptors present in the sera of NYHA functional class IV patients (from 625 to 249 +/- 176 pg/ml, p < 0.001). The results show that TNF-alpha and its soluble receptors are activated in severe CHF. The high concentration of soluble TNF receptors circulating in CHF patients are likely to play a protective role against TNF-alpha biological activity.

Heat shock protein 72 in cardiac and skeletal muscles during hypertension.

In order to elucidate the relationship between hypertension and hypertrophy in the production of heat shock proteins, we studied the induction of the HSP72 synthesis by the heart and gracilis muscles of normo (WKY) and hypertensive (SHR) rats subjected to hyperthermia (42 degrees C +/- 0.5 for 15 min). Two age groups were investigated in each strain: young (2 months, with developing cardiac hypertrophy) and old (18 months, with fully developed chronic cardiac hypertrophy). The gracilis muscle never developed hypertrophy, independently of hypertension or aging. 72 kDa inducible protein was determined by Western blot analysis using a specific monoclonal antibody. We also used a commercial standard, loaded on each blot, to quantitate densitometrically the signal. The heart of young SHR responds to heat shock more than their normotensive age-matched control (298.8 +/- 24.7% vs 88.3 +/- 8.5%, p < 0.001). This response is not maintained during aging as we did not find any significant difference between normo- and hypertensive old rats after exposure to hyperthermia (43.6 +/- 5.3% vs 65.3 +/- 10.4%). Unlike the heart, the gracilis muscle shows a basal spontaneous HSP72 synthesis in both the SHR (71.4 +/- 10.8%) and WKY (40.6 +/- 11.7%) animals. There was a significant increase in HSP72 synthesis in the gracilis muscle of young SHR with respect to their control (186.2 +/- 18.7% vs 115.8 +/- 9.9%, p < 0.02) which was maintained also during aging (171.9 +/- 17.3% vs 95.2 +/- 10.5%, p < 0.01). In conclusion, these data show that hypertension results in an increased synthesis of HSP72 both in cardiac and gracilis muscle in response to heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)