Homocysteine modulates the CD40/CD40L system.

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.

Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans.

OBJECTIVE: Inducible cyclooxygenase (COX-2) catalyzes the first step in prostanoid biosynthesis and is considered a proinflammatory enzyme. COX-2 and type 1 inducible PGE synthase (mPGES-1) have a role in metalloproteinase (MMP) release leading to plaque rupture. In contrast, lipocalin-type PGD synthase (L-PGDS) has been shown to exert antiinflammatory actions. Thus, in this study we investigated whether a shift from a PGDS-oriented to a PGES-oriented profile in arachidonate metabolism leads to inflammatory activation in rupture-prone plaque macrophages. METHODS AND RESULTS: Atherosclerotic plaques were obtained from 60 patients who underwent carotid endarterectomy, symptomatic (n=30) and asymptomatic (n=30) according to evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, mPGES-1, L-PGDS, PPARgamma, IkappaBalpha, NF-kappaB, and MMP-9 by immunocytochemistry, Western blot, reverse-transcriptase polymerase chain reaction, enzyme immunoassay, and zymography. Prostaglandin E2 (PGE2) pathway was significantly prevalent in symptomatic plaques, whereas PGD2 pathway was overexpressed in asymptomatic ones, associated with NF-kappaB inactivation and MMP-9 suppression. In vitro COX-2 inhibition in monocytes was associated with reduced MMP-9 release only when PGD2 pathway overcame PGE2 pathway. CONCLUSIONS: These results suggest that COX-2 may have proinflammatory and antiinflammatory properties as a function of expression of downstream PGH2 isomerases, and that the switch from L-PGDS to mPGES-1 in plaque macrophages is associated with cerebral ischemic syndromes, possibly through MMP-induced plaque rupture.

Urinary excretion of lipoxin A(4) and related compounds: development of new extraction techniques for lipoxins.

LX are tetraene-containing eicosanoids generated by lipoxygenase (LO) transformation of arachidonic acid (Serhan and Romano, 1995). LX possess potent anti-inflammatory activity in vivo, and temporal biosynthesis of LX, concurrent with spontaneous resolution, has been observed during exudate formation (Levy et al, 2001). Limited results are currently available on the involvement of LX in clinical settings. Recently, a rabbit anti-LXA(4) antiserum has been raised to produce an enzyme-linked immunosorbent assay (ELISA) kit for LXA(4) (Levy et al, 1993). Although specific and accurate with isolated cells, this kit has not been tested with complex biological matrix such as urine. Initial attempts to determine urinary excretion of LXA(4) using the LXA(4) ELISA kit were unsuccessful because of high unspecific absorbance readings. In this report, we show that the LXA(4) extraction procedure indicated in the ELISA kit is inadequate for urinary measurements of immunoreactive (i)LXA(4). We present the development of a new extraction technique, more selective for LX, that abolishes background contamination and minimizes the unspecific readings. Using this method, we show for the first time that urine from healthy subjects contain (i)LXA(4) material and identify a urinary tetraene with the physical properties of a LXA(4) metabolite. Although reliable methods have been previously established to quantitate LXA(4) from whole blood (Brezinski et al, 1992), the present extraction technique, which optimizes for LXA(4) recovery from human urine, represents a substantial achievement for LX investigation and may open a new avenue of clinical studies on LXA(4).