Vascular Dysfunction in Aging Mesenteric Arteries of the Rat is Related to Expressions of Inflammatory Regulatory Proteins

Ayotunde Adeagbo1,3, Karni Moshal2,3, Sharon Gordon3, Irving Joshua3. 1The Commonwealth Medical College, Scranton, PA, USA, 2Brown University, Providence, RI, USA, 3University of Louisville, Louisville, KY, USA

Endothelial cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which relax arteries and guard against inflammation (Node et al., 1999; Liu et al., 2005). Although CYP-derived EETs signals through peroxisome proliferator activated receptor (PPARs) and nuclear factor kappa-beta, the mechanism(s) by which EETs-mediated signal cascade causes vascular dysfunction in aging is unclear. Unpublished data from our laboratory show increased oxidative stress (measured as serum 8-epiisoprostanes); reduced CYP2J expression, and decreased synthesis of EETs by aging versus young adult arterial tissues. We hypothesize that arterial dysfunction associated with aging is due to increased oxidative stress, which down-regulates CYP2J2 proteins (therefore EETs synthesis) and PPAR-γ activation in aging versus young adult vasculature. These render vascular tissues amenable to inflammation.

Experiments were performed with arterial tissues obtained from two age categories of male F344BNF1 rats: 4-6 month (Young adults), and 26-30 month (Old). We harvested mesenteric arterial beds from the rats under anesthesia, and perfused (5 ml/min) with physiological salt solution (PSS, 37oC). Changes in perfusion pressure were measured (Statham pressure transducer) and displayed on a Grass polygraph. We also determined arterial cyclooxygenase-2 (COX-2) and membrane prostaglandin E synthase (mPGES) protein expressions by Western blotting, and measured serum levels of PGE2 and C-reactive protein (indices of inflammation) using enzyme immunoassay, and ELISA kits (Cayman Chem. Co, USA), respectively. We also determined serum 8-epi-isoprostanes levels as an index of oxidative stress.

Arterial relaxations elicited by acetylcholine (ACH) are concentration-dependently attenuated by an epoxygenase-selective blocker, PPOH (10-50 μM) suggesting that CYP epoxygenases form acetylcholine-releasable products from arachidonic acid. Such ACH-elicited relaxations decrease significantly in 26-30 month old versus 4-6 (Young adult) month old F344BNF1 rats. Arterial microsomes formed EETs and DHETs (corresponding diols) from 14[C]-AA in an NAD(P)H-dependent, and PPOH-inhibitable manner. Microsomes from old rats formed significantly (P<0.05) less EETs/DHETs versus Young adults microsomes. However, serum PGE2, and C-reactive protein levels as well as 8-Epi-isoprostanes levels increased with age. CYP2J2 and PPAR-gamma protein expressions were significantly decreased; COX-2 and mPGES protein expressions were significantly increased in arteries of 26-30 versus 4-6 month old rats.

We conclude: (1) A CYP2J arachidonate product contributes to mesenteric arterial relaxation evoked by acetylcholine, and the formation of the releasable product is significantly reduced at old age. (2) The significant increases in COX-2, and mPGES protein expressions, and the consequent enhanced serum PGE2 levels coupled with increased serum levels of 8-epi-isoprostanes and C-reactive protein are compatible with development of arterial inflammation.

Node, K., Huo, Y, Ruan, X. et al (1999). Antiinflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science, 285; 1276-1279.

Liu, Y., Zhang, Y., Schmelzer, K et al (2005). The anti-inflammatory effect of laminar flow – the role of PPAR-gamma, epoxyeicosatrienoic acids, and soluble epoxide hydrolase. Proc. Natl. Acad. Sci., 102; 16747-16752.