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.

Aging is characterized by a profound reduction in anti-inflammatory lipoxin A4 levels.

Ongoing low-grade chronic inflammation represents a pathogenetic background for age-related diseases. In this report, we tested the hypothesis that endogenous anti-inflammatory mechanisms may become less efficient with age, resulting in increased susceptibility to inflammatory disorders. Using previously validated ELISA assays, we evaluated urinary levels of the anti-inflammatory, pro-resolution, arachidonic acid (AA) metabolite, lipoxin (LX)A(4) and of the pro-inflammatory cysteinyl leukotrienes (cysLTs) in volunteers aged from 26 to over 100 years. (i) LXA(4) excretion was decreased in elderly people, resulting in a profound unbalance of the LXA(4)/cysLTs ratio, which may be considered an index of the endogenous anti-inflammatory potential. A significant inverse correlation was denoted between age and the LXA(4)/cysLTs ratio (rho = -0.41, P = 0.0026). We conclude that aging is associated with a switch in arachidonic acid metabolism that prevents formation of key 'stop signals' of the inflammatory reaction. This may contribute to promote the development of disease in elderly.

Increased circulating Interleukin-18 levels in centenarians with no signs of vascular disease: another paradox of longevity?

Interleukin (IL)-18 is highly expressed in macrophages from human atherosclerotic plaques, suggesting its involvement in ischemic syndromes. We evaluated IL-18 and IL-18 binding protein (BP) in healthy centenarians, as longevity is characterized by a reduced incidence of ischemic events. For comparison, patients with chronic ischemic syndromes (CIS) were evaluated. Serum IL-18 and IL-18BP levels were measured by non-cross-reacting ELISA in 16 healthy centenarians and in two age-control populations, each of 18 healthy individuals aged 55.9+/-1.43 and 74.3+/-1.35, respectively, as well as in 23 CIS patients, and another cohort of 23 healthy subjects that were age- and sex-matched with CIS patients. Centenarians displayed significantly higher total IL-18 serum levels compared to each control group. Elevated IL-18 levels were also present in CIS patients. However, centenarians had a significant higher level of IL-18BP compared to the cohort of 23 controls (P=0.0014), and compared to CIS patients (P=0.043); as a result centenarians exhibited a lower level of free IL-18 than CIS patients. The present results indicate that quenching of IL-18 by IL-18BP may explain the apparent paradox of elevated serum IL-18 with no vascular signs in centenarians.

Physical exercise increases urinary excretion of lipoxin A4 and related compounds.

Lipoxins (LX) are lipoxygenase-derived eicosanoids with potent anti-inflammatory activities and vascular bed-dependent vasodilatory actions. LX can be formed in vitro and in vivo in a number of conditions, and we have reported that immunoreactive LXA(4) (iLXA(4)) is physiologically excreted with human urine. Using a recently developed LX extraction method coupled to an ELISA, we examined whether iLXA(4) excretion was modified by strenuous exercise, which is known to trigger potential LX-forming events. Maximal exertion significantly increased iLXA(4) urinary excretion in nine healthy volunteers (0.061 +/- 0.023 vs. 0.113 +/- 0.057 ng/mg creatinine; P = 0.028). iLXA(4) levels returned to baseline after 6 h and increased, although at a smaller extent, after 24 h. A significant correlation (r = 0.988) was denoted between iLXA(4) ELISA measurements and reversed-phase high-performance liquid chromatography quantitation of a previously described urinary tetraene, confirming its LXA(4)-related nature. These findings show for the first time that an increase in excretion of LXA(4)-related compounds can be observed in response to strenuous exercise. This may be the reflection of an enhanced LX biosynthesis, which may represent a safeguard mechanism that keeps the inflammatory reaction triggered by physical stress under control.

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).