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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 3, 2002 as doi:10.1096/fj.02-0543fje. |
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,2
Department of Clinical and Experimental Medicine, Institute of Pharmacology and Interdisciplinary Centre for the Study of Inflammation, University of Ferrara, Ferrara, Italy;
* Department of Clinical Medicine and Immunological Sciences, Clinical Immunology Division, University of Siena, Siena, Italy;
Department of Pharmacological Sciences, University of Milano, Milano, Italy;
Department of Surgery and Bioengineering, Section of Thoracic and Cardiovascular Surgery, University of Siena, Siena Italy; and
Department of Pharmacology, Gazi University, Medical Faculty, Ankara, Turkey
3Correspondence: Department of Clinical Medicine and Immunological Sciences, Clinical Immunology Division, University of Siena, Policlinico Le Scotte, 53100 Siena, Italy. E-mail: laghi{at}unisi.it
SPECIFIC AIMS
Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Proinflammatory cytokines (e.g., TNF-
and IL-6) may play a pathogenic role and contribute to progression of left ventricle dysfunction. In CHF patients, peripheral blood mononuclear cells (PBMC) produce great amounts of TNF- and IL-6, indicating that circulating cells are activated and could mirror changes occurring in inflammatory cells infiltrating the failing heart. Adenosine is an endogenous regulatory metabolite acting through specific membrane receptors (the A1, A2A, A2B, and A3 subtypes). The A2A receptor plays multiple roles in the cardiovascular system, including vasodilatation, neoangiogenesis, inhibition of production, and release of proinflammatory cytokines from immune/inflammatory cells. Since in cardiovascular diseases, human circulating cells may express the same alterations occurring in the heart, the present study was designed to monitor adenosine plasma levels and the status of the A2A receptor in the circulating cells and explanted hearts of 40 patients (33 men and 7 women) with terminal CHF referred for cardiac transplantation compared with age-matched healthy subjects. We wanted to assess whether 1) there are any changes of peripheral A2A receptor parameters in CHF patients; 2) these changes mirror receptor changes occurring in the failing myocardium; and 3) in a cohort of 6 patients followed longitudinally, changes of peripheral A2A receptors normalize after heart transplantation.
PRINCIPAL FINDINGS
1. CHF patients show elevated adenosine plasma levels and up-regulated A2A adenosine receptors in peripheral circulating cells
In 39 CHF patients, plasma adenosine concentrations were increased with respect to 32 control subjects (310.5±42.1 vs. 216.7±25.7 nmol/L, P<0.001). Patients showed increased TNF- and IL-6 plasma levels (data not shown). In patients’ lymphocytes, binding studies with the selective A2A receptor ligand [3H]-ZM 241385 revealed a significant increase of Bmax (indicating increased receptor density) with respect to controls (P<0.01) (Fig. 1
A). Similar data were obtained in neutrophils (P<0.01) (Fig. 1B
). RT-PCR analysis in the PBMC of CHF patients revealed a significant increase of A2A receptor expression compared with control subjects (P<0.05) (data not shown). We measured the ability of the A2A receptor agonist N-ethylcarboxamidoadenosine (NECA) to stimulate adenylyl cyclase (the transduction system associated to this receptor) and cAMP accumulation. In lymphocytes from CHF patients, agonist concentration-response curves were shifted to the left with respect to control subjects, indicating an increased potency of NECA (P<0.01) (data not shown). The NECA EC50 value (i.e., the agonist concentration producing half-maximal effect) was significantly lower in cells of CHF subjects vs. controls (136±11 vs. 248±10 nmol/L for CHF patients and control subjects, respectively, P<0.01). Similar data were obtained in neutrophils (EC50 value=121±11 vs. 252±10 nmol/L in CHF patients and control subjects, respectively, P<0.01). We conclude that in CHF patients, altered coupling of increased receptors to transductional G-proteins contributes to the detected amplification of adenylyl cyclase response.
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2. Up-regulation of A2A receptors in peripheral circulating cells of CHF patients mirrors receptor changes occurring in the failing myocardium
To assess whether changes of peripheral A2A receptors in CHF patients could reflect receptor modifications in the heart of the same individuals, we compared [3H]-ZM 241385 binding kinetics in nine explanted hearts of patients undergoing transplantation with those obtained from control subjects. Binding parameters showed alterations (i.e., increased KD and Bmax values, Fig. 2
) comparable to those observed in corresponding blood peripheral cells, suggesting that the latter reproduce the changes occurring in the disease target organ. Increased A2A receptor density in CHF patients is likely due to increased receptor expression, as suggested by preliminary RT-PCR data (data not shown).
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3. Alterations of peripheral A2A receptors in CHF patients progressively normalize after heart transplantation
In a cohort of CHF patients, peripheral A2A receptors were studied longitudinally as a function of time before and after heart transplantation. Figure 3
shows that in lymphocytes of 6 CHF patients, A2A receptor density gradually returned to normal values within 6 months after transplant. This trend was detected in the neutrophils of the same subjects (data not shown). In the PBMC of four patients (cases 2, 4–6), longitudinal RT-PCR analysis revealed a marked and time-dependent decrease of A2A receptor mRNA (data not shown). Consistent with this, in 19 patients analyzed at a single time point 
12 months after transplant compared with 26 control subjects, adenosine A2A receptor mRNA was still 44 ± 8% of control values (P<0.05). In four of the transplanted subjects, adenosine plasma levels progressively decreased during the first 6 months, although they did not reach the normal range within this period. Basal values before and 6 months after transplant changed as follows: case 1, from 305 to 297 nmol/L; case 2, from 312 to 281 nmol/L; case 3, from 360 to 287 nmol/L; case 4, from 321 to 289 nmol/L.
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CONCLUSIONS AND SIGNIFICANCE
The present study highlights, for the first time, an increase of A2A receptor expression, density, and coupling to adenylyl cyclase in the peripheral circulating cells of end-stage CHF patients compared with control subjects. A concomitant modest increase of adenosine plasma levels (likely due to myocardial ischemia and sympathetic activation) was registered in these patients. In line with previous data suggesting that increased production of endogenous adenosine and/or increased expression/activity of its receptors could be compensatory against CHF (likely through A2A receptor-mediated inhibition of cytokine release from inflammatory cells), we speculate that these changes in CHF may reflect the attempt to potentiate adenosine-mediated cytokine inhibition. Similar changes in A2A receptor binding were observed in explanted hearts of CHF patients undergoing cardiac transplantation. We conclude that in CHF, peripheral blood circulating cells express A2A receptor changes that closely mirror the changes occurring in the disease target organ. A second important finding of the present study is the observation that the up-regulation of A2A receptors in circulating cells progressively normalizes after cardiac transplantation. Plasma adenosine levels showed a trend to a decrease to values within the normal range. Hence, the improvement of cardiac performance (and likely of the neurohumoral milieu) after transplantation is associated with a progressive restoration of a normal adenosinergic system, at least for A2A receptor expression and activity. The present results suggest, for the first time, that the status of the A2A adenosine receptor in peripheral blood and myocardial cells of CHF subjects may be indicative of the degree of activation of inflammatory cells infiltrating the failing myocardium. Moreover, our data showing an up-regulation of the A2A receptor in CHF patients and its normalization after heart transplantation (in parallel with the normalization of hemodynamic conditions) support the importance of this receptor in human diseases characterized by a marked inflammatory/immune component, suggesting it may be possible to manipulate inflammation via pharmacological agents selective for this receptor. Finally, by establishing a correlation between receptors in peripheral circulating cells and the myocardium, the present results open up the possibility of exploiting the measure of A2A receptors as a prognostic/diagnostic marker of heart disease and for monitoring hemodynamic changes and efficacy of pharmacological and nonpharmacological treatments in CHF patients.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0543fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0543fje 
2 These authors contributed equally to this work.
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, n=20) and CHF (
, n=26 for lymphocytes; n=23 for neutrophils) subjects. Inset: Scatchard plots of specific binding for calculation of ligand affinity (KD) and maximal number of binding sites (Bmax). B and F denote bound and free ligand, respectively. Data are the mean of different individual experiments. (*P<0.01, one-way ANOVA followed by Student’s t test).
