Quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling.

Functional proteomic strategies offer unique advantages over current molecular array approaches, as the epitopes identified can directly provide bioactive peptides for investigational and/or translational applications. The vascular endothelium is well suited to proteomic assessment by in vivo phage display, but extensive enrichment and sequencing steps limit its application for high throughput molecular profiling. To overcome these limitations we developed a quantitative PCR (Q-PCR) strategy to allow the rapid quantification of in vivo phage binding. Primers were designed for distinct clones selected from a defined phage pool to probe for age-associated changes in cardiac vascular epitopes. Sensitivity and specificity of the primer sets were tested and confirmed in vitro. Q-PCR quantification of phage in vivo confirmed the preferential homing of all phage clones to the young rather than old cardiac vasculature and demonstrated a close correlation with phage measurements previously determined using traditional bacterial-based titration methods. This Q-PCR approach provides quantification of phage within hours of phage injection and may therefore be used for rapid, high throughput analysis of binding of defined phage sequences both in vivo and in vitro, complementing nonbiased phage approaches for the proteomic mapping of vascular beds and other tissues.

Vascular tenascin-C regulates cardiac endothelial phenotype and neovascularization.

Microenvironmental cues mediate postnatal neovascularization via modulation of endothelial cell and bone marrow-derived endothelial progenitor cell (EPC) activity. Numerous signals regulate the activity of both of these cell types in response to vascular injury, which suggests that parallel mechanisms regulate angiogenesis in the vascular beds of both the heart and bone marrow. To identify mediators of such shared pathways, in vivo bone marrow/cardiac phage display biopanning was performed and led to the identification of tenascin-C as a candidate protein. Functionally, tenascin-C inhibits cardiac endothelial cell spreading and enhances migration in response to angiogenic growth factors. Analysis of human coronary thrombi revealed tenascin-C protein expression colocalized with the endothelial cell/EPC marker Tie-2 in intrathrombi vascular channels. Immunostains in the rodent heart demonstrated that tenascin-C also colocalizes with EPCs homing to sites of cardiac angiogenic induction. To determine the importance of tenascin-C in cardiac neovascularization, we used an established cardiac transplantation model and showed that unlike wild-type mice, tenascin-C-/- mice fail to vascularize cardiac allografts. This demonstrates for the first time that tenascin-C is essential for postnatal cardiac angiogenic function. Together, our data highlight the role of tenascin-C as a microenvironmental regulator of cardiac endothelial/EPC activity.

BDNF-mediated enhancement of inflammation and injury in the aging heart.

Aging is associated with shifts in autocrine and paracrine pathways in the cardiac vasculature that may contribute to the risk of cardiovascular disease in older persons. To elucidate the molecular basis of these changes in vivo, phage-display biopanning of 3- and 18-mo-old mouse hearts was performed that identified peptide epitopes with homology to brain-derived neurotrophic factor (BDNF) in old but not young phage pools. Quantification of cardiac phage binding by titration and immunostaining after injection with BDNF-like phage identified a twofold increased density of the BDNF receptor, truncated Trk B, in the aging hearts. Studies focused on the receptor ligand using a rat model of transient myocardial ischemia revealed increases in cardiac BDNF associated with local mononuclear infiltrates in 24- but not 4-mo-old rats. To investigate these changes, both 4- and 24-mo-old rat hearts were treated with intramyocardial injections of BDNF (or PBS control), demonstrating significant inflammatory increases with activated macrophage (ED1+) in BDNF-treated aging hearts compared with aging controls and similarly treated young hearts. Additional studies with permanent coronary occlusion following intramyocardial growth factor pretreatment revealed that BDNF significantly increased the extent of myocardial injury in older rat hearts (BDNF 35 +/- 10% vs. PBS 16.2 +/- 7.9% left ventricular injury; P < 0.05) without affecting younger hearts (BDNF 15 +/- 5.1% vs. PBS 14.5 +/- 6.0% left ventricular injury). Overall, these studies suggest that age-associated changes in BDNF-Trk B pathways may predispose the aging heart to increased injury after acute myocardial infarction and potentially contribute to the enhanced severity of cardiovascular disease in older individuals.

Phage display identification of age-associated TNFalpha-mediated cardiac oxidative induction.

Age-associated alterations in the actions of tumor necrosis factor-alpha (TNFalpha) in the heart with impaired cardioprotective pathways and enhanced apoptotic induction may contribute to the increased severity of cardiovascular pathology in older persons. To identify the molecular events mediating these changes in the microvasculature of the aging rodent heart, the biochemical properties of in vivo phage-display cyclic peptide cardiac biopanning were studied. Analysis of individual amino acid positions revealed that the center of the peptide motif (amino acid position 4) had a significantly higher frequency of aromatic amino acid side chains in phage homing to the old hearts compared with young controls (18 mo old, 11% vs. 3 mo old, 3%, P < 0.05). This subset of phage motifs revealed an age-associated homology with oxidoreductase enzymes (homology: 18 mo, 7/7; 3 mo, 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 twofold higher density of HNE(+) cells in PBS-treated hearts of old mice (18 mo) compared with young controls (3 mo) (18 mo, 3.2 +/- 2.8 vs. 3 mo, 1.0 +/- 0.9 cells/HPF, P < 0.05). Moreover, intracardiac injection of TNFalpha resulted in a significantly greater increase in HNE staining in the old hearts (18 mo, 16.9 +/- 13.8 vs. 3 mo, 9.1 +/- 6.0 cells/HPF, P < 0.05). Overall, these studies demonstrate that aging-associated alterations in TNFalpha-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.

Senescent impairment in synergistic cytokine pathways that provide rapid cardioprotection in the rat heart.

Pretreatment of rodent hearts with platelet-derived growth factor (PDGF)-AB decreases myocardial injury after coronary occlusion. However, PDGF-AB cardioprotection is diminished in older animals, suggesting that downstream elements mediating and/or synergizing the actions of PDGF-AB may be limited in aging cardiac vasculature. In vitro PDGF-AB induced vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 expression in 4-mo-old rat cardiac endothelial cells, but not in 24-mo-old heart cells. In vivo injection of young hearts with PDGF-AB increased densities of microvessels staining for VEGF and its receptor, Flk-1, and Ang-2 and its receptor, Tie-2, as well as PDGF receptor (PDGFR)-alpha. In older hearts, PDGF-AB-mediated induction was primarily limited to PDGFR-alpha. Studies in a murine cardiac transplantation model demonstrated that synergist interactions of PDGF-AB plus VEGF plus Ang-2 (PVA) provided an immediate restoration of senescent cardiac vascular function. Moreover, PVA injection in young rat hearts, but not PDGF-AB alone or other cytokine combinations, at the time of coronary occlusion suppressed acute myocardial cell death by >50%. However, PVA also reduced the extent of myocardial infarction with an age-associated cardioprotective benefit (4-mo-old with 45% reduction vs. 24-mo-old with 24%; P < 0.05). These studies showed that synergistic cytokine pathways augmenting the actions of PDGF-AB are limited in older hearts, suggesting that strategies based on these interactions may provide age-dependent clinical cardiovascular benefit.

Age-associated impairment in TNF-alpha cardioprotection from myocardial infarction.

Age-associated dysfunction in cardiac microvascular endothelial cells with impaired induction of cardioprotective platelet-derived growth factor (PDGF)-dependent pathways suggests that alterations in critical vascular receptor(s) may contribute to the increased severity of cardiovascular pathology in older persons. In vivo murine phage-display peptide library biopanning revealed a senescent decrease in cardiac microvascular binding of phage epitopes homologous to tumor necrosis factor-alpha (TNF-alpha), suggesting that its receptor(s) may be downregulated in older cardiac endothelial cells. Immunostaining demonstrated that TNF-receptor 1 (TNF-R1) density was significantly lower in the subendocardial endothelium of the aging murine heart. Functional studies confirmed the senescent dysregulation of TNF-alpha receptor pathways, demonstrating that TNF-alpha induced PDGF-B expression in cardiac microvascular endothelial cells of 4-mo-old, but not 24-mo-old, rats. Moreover, TNF-alpha mediated cardioprotective pathways were impaired in the aging heart. In young rat hearts, injection of TNF-alpha significantly reduced the extent of myocardial injury after coronary ligation: TNF-alpha, 7.9 +/- 1.9% left ventricular injury (n = 4) versus PBS, 16.2 +/- 7.9% (n = 10; P < 0.05). The addition of PDGF-AB did not augment the cardioprotective action of TNF-alpha. In myocardial infarctions of older hearts, however, TNF-alpha induced significant postcoronary occlusion mortality (TNF-alpha 80% vs. PBS 0%; n = 10 each, P < 0.05) that was reversed by the coadministration of PDGF-AB. Overall, these studies demonstrate that aging-associated alterations in TNF-alpha receptor cardiac microvascular pathways may contribute to the increased cardiovasular pathology of the aging heart. Strategies targeted at restoring TNF-alpha receptor-mediated expression of PDGF-B may improve cardiac microvascular function and provide novel approaches for treatment and possible prevention of cardiovascular disease in older individuals.