Age-associated alterations in the actions of tumor necrosis factor (TNF) in the heart with impaired cardioprotective pathways and enhanced apoptotic induction may contribute to the increased severity of cardiovascular pathology in older persons. In order to identify the molecular events mediating these changes in the microvasculature of the aging rodent heart, the biochemical properties of in vivo phage-display seven amino acid cyclic peptide cardiac biopanning were studied. Analysis of individual amino acid positions revealed that the center of peptide motif (amino acid position 4) had a significantly higher frequency of aromatic amino acid side chains in phage homing the old hearts compared with young controls (18m-19% vs. 3m-2%, P<0.05). This subset of phage motifs revealed an age-associated homology with oxidoreductase enzymes (homology:18m-7/7; 3m-0/2), suggesting the substrates and/or binding sites of these enzymes are increased in the aging hearts. Immunostaining for the oxidoreductase substrate 4-hydroxy-2-nonenal (HNE), a cardiotoxic lipid peroxidation product, demonstrated a two-fold higher density of HNE(+) cells in PBS treated hearts of old mice (18 m) compared with young controls (3 m) (18m-10.1+/-8.0 cells/HPF vs. 3m. 1.0 +/-.9 cell/HPF, P<0.05). Moreover intracardiac injection of TNF resulted in a significantly greater increase in HNE staining in the old hearts (18m-16.9+/-13.8 cells/HPF vs. 3m - 3.2+/-2.8 cell/HPF, P<0.05). Overall, these studies demonstrate that aging-associated alterations in TNF mediated pathways with induction of reactive oxidative species and changes in vascular surface binding sites may contribute mechanistically to the increased cardiovascular pathology of the aging heart.