Phage display identification of age-associated TNF{alpha}-mediated cardiac oxidative induction

Jay M. Edelberg, Alvin Wong, Jacquelyne M. Holm, Munira Xaymardan, Inga Duignan, Andrew Chin, Jorge R. Kizer and Dongqing Cai

Departments of Medicine and Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10021


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Age-associated alterations in the actions of tumor necrosis factor-{alpha} (TNF{alpha}) 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 TNF{alpha} 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 TNF{alpha}-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.

aging; heart; endothelial; functional genomics; oxidation


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AGE-ASSOCIATED CHANGES in the cardiovascular system may predispose older individuals to increased vascular pathology. Previous studies have demonstrated that altered regulation and dysfunction of vascular endothelial cells is linked to worsened clinical outcomes in cardiovascular disease (25, 30). Recently, we demonstrated that age-associated impairment of the tumor necrosis factor-{alpha} (TNF{alpha}) receptor pathways in cardiac microvascular endothelial cells contributes to cardiac microvascular dysfunction with enhanced apoptotic induction and suppressed angiogenic pathways in the aging rodent heart (5). Indeed, delivery of TNF{alpha} is cardioprotective in the young rat heart but results in significant mortality after myocardial infarction in older rats (5). Defining the molecular events mediating this shift in TNF{alpha} function in the aging heart may facilitate the development of approaches to decrease the impact of cardiovascular disease in the geriatric population.

We hypothesized that the senescent alterations in TNF receptor patterning are associated with biochemical changes in the cardiac vascular endothelial cells that may be related to the shift in TNF{alpha} signaling pathways in the aging heart. Specifically we sought to define pathophysiological pathways mediated by senescent shift in the cardiac actions of TNF{alpha} through an analysis of the molecular phenotypic changes in the aging cardiac microvasculature. Based on the role of the in vivo phage display motifs to identify the age-associated decline in TNF-R1 subpopulations, we hypothesized that further characterization of the peptide motifs would provide important insights into the potential pathophysiology of the senescent shift in TNF{alpha} pathways. To elucidate such mechanistic links to the age-associated changes in TNF{alpha} pathways, we performed a biochemical assessment of in vivo phage-display biopanning studies in young and old rodent hearts (Fig. 1).



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Fig. 1. Phage-display biopanning assessment of age-associated changes in cardiac microvasculature. Based on previous direct homology analysis revealing changes in tumor necrosis factor-{alpha} (TNF{alpha}) receptors (5), an analysis of the biochemical properties of the motifs was employed to define additional pools of peptides for homology analysis to identify molecular and cellular pathways that may provide the mechanistic links to the shift in TNF{alpha} actions in the aging heart.

 
Here we report the results of a biochemical analysis of in vivo phage-display biopanning studies in young and old rodent hearts that led to the identification of an age-associated increase of TNF{alpha}-induced cardiac lipid peroxidation that may contribute to the alterations in TNF{alpha} pathways in the older heart.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In vivo phage display and biopanning.
The age-associated changes in cardiac microvascular surface binding epitope distribution were probed by in vivo phage-display biopanning with a cyclic peptide pSKAN phagemid library (6 amino acid variable; ~107 total complexity; MoBioTech, Goettingen, Germany), as previously described (5). Young adult (3 mo old) and aging (18 mo old) C57B61/L mice (National Institute on Aging Colony, maintained by Harlan Sprague Dawley) were anaesthetized with Avertin (0.015 ml/g) and injected with phage peptide library phage (1012 CFU/200 µl PBS) via the tail veins. Four minutes after injection, the mice were killed, the hearts were explanted, and the phage recovered with WK6{lambda}mutS E. coli. Age-specific phage pools were amplified and titrated for two additional rounds of biopanning enrichment. The phagemid DNA of the resultant clones was sequenced, and translated amino acid motifs were determined from all open-reading frame variable regions (n = 64, 3 mo old; n = 61, 18 mo old).

Biochemical analysis of phage peptide motifs.
The peptide motifs isolated from the serial cardiac biopanning procedure were molecularly characterized by three approaches: 1) the theoretical isoelectric pH values (pI values) of the variable regions were calculated using PEPTIDEMASS as previously described (31); 2) the overall acid-base distribution of the peptide motifs was determined by the net balance of the amino acid side chains (acidic –1; basic +1; neutral 0); and 3) the properties of the individual amino acid positions (16) of the variable motifs were quantified by side chain characteristics (hydrophobic, hydrophilic, aliphatic, aliphatic hydroxyl, acidic, basic, amide, aromatic, sulfuric, and cyclic). Based on statistically significant differences in the distribution of aromatic amino acid side chains at position 4 in the old vs. young phage pools, the translated motifs were analyzed for homology (FASTA3) to previously defined proteins. Protein(s) with homology to a significant fraction of the differentially distributed motifs (E < 1, ≥1 clone) was analyzed as described below.

Immunohistochemical analysis of in vivo response to TNF{alpha}.
Based on senescent differential distribution patterns with homology of oxidoreductase species, the potential age-related differences of reactive oxygen species (ROS) products in myocardial response to TNF{alpha} were measured. Briefly, sets of 3- and 18-mo-old mice were anesthetized and underwent left intercostal thoracotomy. After identifying the left anterior descending artery (LAD), 20 ng of TNF{alpha} in 50 µl PBS or PBS alone was injected through a 30-gauge needle using a 250-µl Hamilton syringe. Two injections (25 µl/injection, 2 mm apart) were made at the mid left ventricular anterior wall. The chest wall was then closed, the lungs inflated, the mice were extubated, and the tracheotomy was closed. Twenty-four hours postinjection, the mice were once again anesthetized, then euthanized, and their hearts were explanted. The tissue sections were stained with a polyclonal antibody to 4-hydroxy-2-nonenal (HNE; Alexis Biochemicals, San Diego, CA) developed with Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA), and the number of positive staining cells per high-power field (HPF) was quantified in eight independent fields of the anterior wall of the murine hearts (n = 3 hearts, each set).

To isolate the senescent alterations in cardiac-induced vascular function from the role of aging on the endogenous cardiac environment, we employed a neonatal cardiac allograft model in young and old syngeneic host mice. In this model, allograft neovascularization is mediated by host endothelial cells recruited into the donor hearts that recapitulate the cardiac myocyte-endothelial cell communication in vivo (11, 32). Sets of 3- and 18-mo-old C57B61/L mice were treated with subcutaneous pinnal cytokine injection (20 mg TNF{alpha} or PBS) 24 h prior to syngeneic neonatal heart transplantation (n = 3, each set). The mice were killed 24 h after transplantation, and the pinnal allografts were explanted, sectioned, and immunostained for HNE as described above.

Statistical analysis.
Comparisons of categorical variables were performed using Fisher’s exact test. For both ordinal and nonnormally distributed continuous variables, the Wilcoxon rank-sum test was applied. Statistical significance was defined by a two-tailed P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Age-associated phage peptide motif characterization.
Analysis of the overall properties of the variable motifs binding to the young and old hearts were similar. The isoelectric pH distributions of phage motifs homing to the young and old hearts were comparable (Fig. 2A). In addition, the calculated net charges of peptide sequences were also similar in the two phage pools, ranging from a net of two basic amino acids to four side chains and favoring a single net positive charge in the phage motif (Fig. 2B).



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Fig. 2. Biochemical analysis of phage peptide motifs distribution patterns. A: scatter plot of isoelectric point distribution of phage peptide motifs. Isoelectric points were calculated for the variable motifs of phage clones amino acid sequences after in vivo biopanning of the heats of 3- and 18-mo-old mice and revealed similar distribution patterns with no statistical differences. B: histogram plot of acid-base balance of distribution of phage peptide motifs. The net balance of each phage peptide motif was calculated (acid –1, base +1, neutral 0). The young and old cardiac phage motifs had distribution means of +1 charge with no significant age-associated differences in acid-base patterning.

 
Based on the similarities in the overall characteristics, an analysis of the composition of the amino acid distributions at the individual positions within the variable regions was performed. Significant differences in the side chain characteristics were limited to the center of the constrained peptide motif, amino acid position 4. The phage isolated from the old hearts had a lower percentage of hydrophilic side chains at position 4 compared with the phage homing to young hearts (Fig. 3). Conversely, there was a statistically significant increase in the number of hydrophobic and aromatic amino acids in the old murine heart phage pool.



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Fig. 3. Age-associated differences in amino acid properties at peptide motif position 4. Percentages of hydrophilic, hydrophobic, and aromatic amino acids at the fourth position of the motif variable insert of the phage clones resulting from the in vivo biopanning of the young and old murine hearts. Distribution patterns of amino acids at other positions revealed no statistical differences between the motifs from the young and old hearts. *P < 0.05 3-mo-old vs. 18-mo-old.

 
Age-based differential distribution of oxidoreductase species homology.
Homology analyses were performed for the motifs with aromatic side chains at variable position 4. These studies demonstrated that the differentially distributed aromatic motifs revealed repetitive homology with oxidoreductase enzymes in all of the peptides in the older cardiac pool (6, 7, 9, 13, 16, 24, 26), but not in the two motifs of the young hearts (Fig. 4). This motif analysis did not identify a common enzyme, with oxidoreductases identified from both prokaryotic and eukaryotic species, suggesting that a common substrate/binding site of oxidoreductases may be more prevalent in the aging hearts



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Fig. 4. The age-associated increase in cardiac homing phage peptide motifs with aromatic amino acids at center of cyclic peptides (position 4) revealed significant homology to oxidoreductase enzymes, highlighted in yellow and outlined by dashed lines in representative examples including the following: {Psi}O145, aldehyde ferredoxin oxidoreductase (A); {Psi}O90, NADPH-dependent thioredoxin reductase (B); and {Psi}O11, extradiol dioxygenase (C). D: the sequence, name, and homology score of the species of oxidoreductase identified by analysis of the phage homing to the 18-mo-old mouse hearts. The phage subpopulation homing to the 3-mo-old heart did not have homology to any oxidoreductase species, and most homologous protein is listed.

 
TNF{alpha} induces age-associated differential cardiac reactive oxidative species.
Based on previous studies revealing that cardiac oxidoreductases recognize the toxic oxidized lipid product HNE (28, 29), sections of young and old murine hearts were analyzed for HNE. Immunohistochemistry demonstrated that HNE(+) staining was significantly higher in the 18-mo-old, compared with 3-mo-old, control hearts (Fig. 5, A and B). TNF{alpha} specifically increased HNE in the older cardiac tissue to a significantly higher density compared with both control old hearts and the TNF{alpha}-treated young hearts, with a significantly lower increase the density of HNE cellular staining in the young heart.



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Fig. 5. Age-associated TNF{alpha} induction of cardiotoxic lipid peroxidation. A: representative immunostaining for HNE (arrows indicate positive staining) in sections of 3- and 18-mo-old murine hearts harvested 24 h after intramyocardial treatment with TNF{alpha} or control (PBS). Bar = 10 µm. B: density of HNE(+) cells in the cardiac tissues (n = 8 fields/heart, 3 hearts/sample set). *P < 0.05. C: representative immunostaining for HNE (arrows indicate positive staining) in sections neonatal cardiac allografts (asterisks in C indicate allograft chamber) transplanted into the pinnae of 3- and 18-mo-old mice pretreated 24 h prior to transplantation with subcutaneous injections of TNF{alpha} or control (PBS) and harvested 24 h posttransplantation. Bar = 100 µm.

 
To control for the senescent changes in the cardiac microenvironment and specifically study the role of aging in TNF{alpha}-induced cardiac vascular function, a syngeneic neonatal murine cardiac allograft-pinnal transplant model was employed. This model recapitulates cardiac microvascular communication pathways in the organ bed-specific regulation of endothelial cells recruited from host vascular beds to facilitate the in vivo study of endothelial cells of different age host mice in a controlled cardiac microenvironment (1, 11). Immunostains of the donor cardiac allografts in 18-mo-old hosts that were treated with TNF{alpha} prior to heart transplantation revealed more widespread HNE in the engrafted cardiac tissue compared with transplants in younger mice or older mice pretreated with PBS (Fig. 5C).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TNF{alpha} mediates a diverse array of both beneficial and deleterious molecular and cellular cardiovascular responses, ranging from mediating protective cardiac preconditioning to inducing myocyte and endothelial cell apoptosis (4, 8, 10, 12, 20, 27). Aging can shift the balance to enhance cardiac pathogenesis (5). The pathways governing these complex multicellular responses as well as TNF{alpha}-mediated pathophysiology in the heart are complex. Our present functional genomic/proteomic studies identified age-associated changes in phage motif characteristics that led to the identification of an increase in TNF{alpha} induction of ROS in the aging heart which may contribute to the senescent switch in the actions of TNF{alpha} from cardioprotection to pathogenesis.

The age-associated shift to ROS pathways may contribute to the increase in apoptosis observed in vitro as well as in vivo in aging vascular cells. Previous studies have demonstrated that TNF{alpha} signaling pathways are altered with aging, with enhanced apoptotic induction in endothelial cells (5, 17). These findings, coupled with the documented role of ROS in TNF{alpha} signaling (2, 14, 15, 19), suggest that the senescent shift to ROS production may underlie the vascular dysfunction and myocardial impairment observed in aging, with a loss in cardioprotective TNF receptor endothelial cells. Moreover, the enhanced cardiac microvascular oxidation with formation of toxic oxidized lipid products may directly contribute to the increased TNF{alpha}-induced apoptosis in aging endothelial cells (17) and lead to cardiac myocyte injury and additional ROS induction in the heart. Furthermore, age-associated chronic increases in systemic levels of TNF{alpha} (3, 21) may also promote cardiac oxidation and compound vascular dysfunction with loss of cardioprotective microvascular endothelial cells. Elucidation of the molecular pathways mediating the balance of TNF{alpha}-induced ROS production and anti-apoptotic signaling mechanisms in the cardiac microvasculature may provide an important foundation for developing novel cardiovascular therapies.

The application of in vivo phage display for characterizing changes in the biochemical properties offers a novel tool to study the intact vasculature. Previous studies have employed phage-based biopanning assays to define vascular bed-specific motifs (22) and have established the foundation for new therapies based on the bioactivity of individual peptide structures (18, 23). These studies have been primarily based on the identification of commonly expressed motifs and homologous peptides as was employed in defining the age-associated changes in TNF{alpha} receptors and function in the rodent heart (5). The present studies reveal that analysis of the properties of the peptides of the biopanning studies also has utility in characterizing the alterations in physiological/pathophysiological pathways. Indeed, our results of composite analysis of the phage motifs isolated from the young and old hearts suggested that the overall biochemical properties of the cardiac homing peptides, as well as their vascular surface binding sites, were similar. Rather, it was the study of the distribution of the amino acid side chains at the various positions that led to the identification of a statistically significant subpopulation of phage motifs to provide insight into the age-associated changes in TNF{alpha}-mediated vascular activity in the local microvascular environment, which may also reflect changes in the surrounding cells, such as cardiac myocytes, to further impact senescent cardiac vascular function.

The greater number of aromatic amino acids in the center of the cyclic peptide directed our assessment of this subpopulation of cardiac homing phage with homology to a wide array of oxidoreductases from multiple species, suggesting that common binding sites of these enzymes, including substrates but not necessarily the enzymes themselves, may be more prevalent in the aging heart. As the ROS product HNE is a major substrate of cardiac oxidoreductases, our studies were able to identify the age-associated increases in the total magnitude of TNF{alpha}-induced HNE that may contribute to the TNF{alpha}-associated pathology in the older cardiac microvasculature. Indeed, the findings in the pinnal model reveal that the actions of TNF{alpha} were mediated through treatment of the engraftment site, as opposed to the cardiac tissue directly. Together, these results suggest that age-associated changes in the vasculature supplying the transplanted neonatal hearts as well as the endogenous cardiac microvasculature translated into increased cardiac oxidative stress in the older animals. Although further research is required to elucidate the mechanism governing the proinflammatory increase in the cardiac actions of TNF{alpha}, the present studies suggest that phage display can be employed as an in vivo "biochemical library" to study the properties of various physiological and pathophysiological processes. Indeed, analysis of a larger pool of cardiac vascular homing phage motifs utilizing both common homology algorithms as well as biochemical property based approaches will likely provide important additional understandings of the age-associated changes in cardiac microvascular function, including the senescent enhancement in oxidative pathways.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by grants to J. M. Edelberg from the American Federation for Aging Research, Beeson Physician Faculty Scholar in Aging Research, and by the National Institute on Aging Grants AG-20320 and AG-20918.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. M. Edelberg, Weill Medical College of Cornell Univ., 520 East 70th St., A352, New York NY 10021 (E-mail: jme2002{at}med.cornell.edu).

10.1152/physiolgenomics.00161.2003.


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