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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:1240.)
© 2004 American Heart Association, Inc.

Vascular Biology

Enhanced P2X₇ Activity in Human Fibroblasts From Diabetic Patients

A Possible Pathogenetic Mechanism for Vascular Damage in Diabetes

Anna Solini; Paola Chiozzi; Anna Morelli; Elena Adinolfi; Roberta Rizzo; Olavio R. Baricordi; Francesco Di Virgilio

From the Department of Internal Medicine (A.S.), University of Pisa, Italy; the Department of Experimental and Diagnostic Medicine (P.C., A.M., E.A., R.R., O.R.B., F.D.V.), University of Ferrara, Italy; and the Interdisciplinary Center for the Study of Inflammation (F.D.V.), University of Ferrara, Italy.

Reprint requests to Dr Francesco Di Virgilio, Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Via Borsari 46, I-44100 Ferrara, Italy. E-mail fdv{at}unife.it

Objective— We have investigated expression and function of the P2X₇ receptor in fibroblasts from healthy subjects and patients with type 2 diabetes.

Methods and Results— Fibroblasts were isolated from skin biopsies. P2X₇ receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X₇-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3x as much ATP into the supernatants compared with fibroblasts from healthy subjects.

Conclusions— We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X₇ reactivity.

Fibroblasts from type 2 diabetic subjects exhibit a higher level of P2X₇ receptor activity compared with fibroblasts from healthy subjects, as witnessed by a larger ATP-dependent release of IL-6 and fibronectin and a higher susceptibility to ATP-dependent cytotoxicity. In addition, fibroblasts from diabetic subjects also secrete a larger amount of ATP compared with controls.

Key Words: P2 receptors • fibroblasts • atherosclerosis • cytokines • diabetes • apoptosis

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