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CLINICAL RESEARCH

Homocysteine Modulates the CD40/CD40L System

Cesaria Prontera, PhD^_*, Nicola Martelli, Tech, Virgilio Evangelista, MD, Etrusca D'Urbano, Tech, Stefano Manarini, Tech, Antonio Recchiuti, PhD^_*, Alfredo Dragani, MD, Cecilia Passeri, MD, Giovanni Davì, MD^_* and Mario Romano, MD^,_*

^_* Department of Medicine, Gabriele d'Annunzio University Foundation, Chieti-Pescara, Italy
Laboratory of Vascular Biology and Pharmacology, Consorzio Mario Negri Sud, San Maria Imbaro, Italy
Department of Biomedical Sciences and Aging Research Center, Gabriele d'Annunzio University Foundation, Chieti, Italy
Civil Hospital, Pescara, Italy

Manuscript received January 31, 2006; revised manuscript received December 20, 2006, accepted February 9, 2007.

^_* Reprint requests and correspondence: Dr. Mario Romano, Dipartimento di Scienze Biomediche, Ce.S.I., Universitá G. D'Annunzio, 66013 Chieti, Italy. (Email: mromano{at}unich.it).

Objectives: This study evaluated the impact of hyperhomocysteinemia (HHcy) on the CD40/CD40 ligand (CD40L) dyad in vivo and in vitro.

Background: Hyperhomocysteinemia is associated with an increased incidence of atherothrombosis, although the molecular mechanisms of this association are incompletely defined. The CD40L pair triggers inflammatory signals in cells of the vascular wall, representing a major pathogenetic pathway of atherosclerosis.

Methods: We used a commercially available enzyme-linked immunosorbent assay kit to evaluate circulating levels of soluble (s) CD40L in 24 patients with HHcy and 24 healthy subjects. We also used real-time polymerase chain reaction and flow cytometry to determine expression levels of CD40 and vascular cell adhesion molecule (VCAM)-1 in human umbilical vein endothelial cells (HUVECs) and of CD40L in human platelets.

Results: The sCD40L levels were significantly increased in HHcy patients (median [interquartile range] 8.0 [0.7 to 10.5] ng/ml vs. 2.1 [1.9 to 2.3] ng/ml, p = 0.0001). Positive correlations were noted between log sCD40L and log homocysteine (Hcy) (R = 0.68, p < 0.0001) or log sVCAM-1 (R = 0.41, p < 0.005). Homocysteine significantly stimulated CD40 mRNA expression in HUVECs (p = 0.033). Consistently, 24-h exposure to Hcy increased the percentage of CD40-expressing cells (p = 0.00025). Homocysteine also significantly enhanced CD40L expression in platelets (p = 0.025) to a comparable extent as that of thrombin. Notably, Hcy increased VCAM-1 protein expression induced by CD40L in HUVECs (p = 0.0046).

Conclusions: The present results uncover a potential molecular target of Hcy, namely the CD40/CD40L dyad. Collectively, they indicate that upregulation of CD40/CD40L signaling may represent a link between HHcy and an increased risk of cardiovascular disease.

Abbreviations and Acronyms   BSA = bovine serum albumin   CD40L = CD40 ligand   EDTA = ethylenediaminetetraacetic acid   Hcy = homocysteine   HEPES = N-2 hydroxyethyl piperazine-N 1-2-ethanesulfonic acid   HHcy = hyperhomocysteinemia   HUVECs = human umbilical vein endothelial cells   Ig = immunoglobulin   MFI = mean fluorescence intensity   MTHFR = 5,10 methylene tetrahydrofolate reductase   NF = nuclear factor   PBS = phosphate-buffered saline   s = soluble   VCAM = vascular cell adhesion molecule

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