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Aging and Cardiovascular Angiogenesis Models

Andrew Chin, Jacquelyne M. Holm, Inga J. Duignan, and Jay M. Edelberg

The development of new strategies to reduce the impact of heart disease in the aging population is an increasingly important public health issue. Indeed, although present clinical therapies have reduced the overall prevalence of cardiovascular disease (Hansson et al., 1999; Yusuf et al., 2000; Volpe et al., 2005), myocardial infarction and congestive heart failure remain the most significant cause of morbidity and mortality in older individuals (Black, 2003; Oxenham et al., 2003; Stewart et al., 2003). To this end, recent studies, primarily in rodent models, suggest that impairment in cardiac angiogenesis may predispose the aging heart to more severe patho-physiology and lead to the worst clinical outcomes observed in the geriatric population. This chapter aims to review integrated methodological approaches to aid in the investigation of senescent cardiac angiogenesis in order to facilitate further studies into the basic biological changes underlying the age-associated alterations in cardiac angiogenic regulation and potentially enhance the preclinical testing of novel therapeutic approaches based on such discoveries.

Introduction

Previous studies have identified age-associated changes in the heart that may serve as potential targets to reverse the geriatric predisposition to more severe cardiovascular pathology. Specifically, age-associated changes in cardiac growth factor expression, vascular wall composition, free radical production, and shifts in apoptotic pathways may contribute to increased cardiac damage after coronary artery occlusion in the older heart. Indeed, experimental strategies based on these findings may offer potential therapeutic approaches to diminish the overall impact of cardiovascular disease in the aging population.

To fully understand the impact of the age-related changes in cardiac angiogenesis, in vivo models are required. It is essential that preclinical development of therapies based on these biological changes with aging be fully tested in age-appropriate models of cardiovascular disease in order to increase the translational potential of treatment of older persons. Based on the importance of rodent models in the basic investigation of the biology of aging in the cardiovascular system, as well as the utility of mice and, to a lesser extent, rats in models of human pathophysiology, this review will aim to summarize in vivo models of cardiac angiogenesis, including models of cardiovascular disease and interven-tional protocols.

Cardiac Angiogenesis: Identification of Biological Changes

Aging is associated with significant alterations at multiple levels of cell regulation, including growth factor expression, receptor signaling, and cell migration. Previous studies employing a spectrum of molecular, cellular, and physiological approaches have demonstrated that alterations in vascular growth factor pathways (Edelberg et al., 2002b; Xaymardan et al., 2004b) result in endothelial cellular dysfunction (Rivard et al., 1999; Cai et al., 2003) and impaired angiogenesis (Nakae et al., 2000; Shimada et al., 2004), which may underlie the senescent predisposition to increased vascular disease in the aging heart. To this end, endothelial dysfunction is one of the most important risk factors for cardiovascular disease in the general population (Widlansky et al., 2003), and its impact is heightened with age. Physiologically, vascular endothelial growth factor-nitric oxide (NO) pathways, which play a central role in endothelial-mediated vasodi-lation, are impaired with aging (Berkowitz et al., 2003) and may result in increased myocardial injury after coronary occlusion compared to younger hearts. In the heart episodes of cardiac tissue hypoxia caused by transient coronary occlusion induce vasodilatory actions that result in cardioprotection in the young rodent heart (Rochetaing et al., 2003). In the aged heart, this ischemic preconditioning can be significantly depressed (Abete et al., 1997; Tani et al., 1999; Fenton et al., 2000). Beyond changes in vascular endothelial growth factor (VEGF) (Rivard et al., 1999), experimental models have demonstrated impairments in the expression and function of angiogenic factors including basic fibro-blast growth factor (b-FGF) (Augustin-Voss et al., 1993;

Handbook of Models for Human Aging

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Garfinkel et al., 1996), transforming growth factor beta (TGF-£) (Reed et al., 1998), and platelet-derived growth factor (PDGF) (Sarzani et al., 1991; Edelberg et al., 2002b; Cai et al., 2003) with aging, suggesting that strategies directed at these and other senescent changes may restore angiogenic pathways in the aging heart and may have clinical utility in conjunction with present reperfusion therapies.

Candidate Testing

Angiogenesis is a dynamic process that requires inductive models to investigate the biological significance of potential regulatory mechanisms. Hence, although characterization of age-related differences in angiogenic components can facilitate the discovery of the potential mechanisms underlying the senescent impairment in cardiac angiogenesis, the in vivo significance of these findings can only be elucidated through interventions to modulate angiogenic induction. To this end, models testing angiogenic growth factors such as VEGF (Ferrara, 2001), bFGF (Yanagisawa-Miwa et al., 1992; Harada et al., 1994), and PDGF (Edelberg et al., 2002b) have been shown to induce angiogenesis in the aging vasculature. In the heart, molecular and protein, as well as cell-based approaches, have been studied in models aimed at determining the significance of different pathways in senescent cardiac angiogenic impairment.

As investigations continue to develop the understanding of the biological basis of the changes in senescent cardiac angiogenic impairment, studies that employ established in vivo approaches to assess the functional impact of age-associated changes in elements and pathways will continue to be required. Unfortunately, the modern tools of genetic engineering do not readily lend themselves to high throughput investigations of aging and cardiac angiogenesis. Indeed, the potential combination of allele targeting and colony aging negates the power of knockout mice as an initial tool to investigate the significance of genes associated with the senescent impairment in cardiac angiogenesis. Thus in vivo models employing wild type aged animals can allow the best initial assessment of the potential contribution to candidate genes, proteins, and pathways associated with the biological changes of angiogenesis in the aging heart.

Cardiac Allograft Assay

In order to more rapidly screen the functional significance of age-associated changes in cardiac angiogenic pathways without the requirement of direct cardiac targeting, a cardiac allograft model of angiogenesis is employed (see Figure 78.1). This model, in which neonatal cardiac tissue is engrafted into the pinnal tissue of various age syngeneic mice, allows for the isolation of age on the angiogenic vasculature (Edelberg et al. , 2002b; Edelberg

Viable Non-Viable

Figure 78.1 This cartoon illustrates the neonatal cardiac allograft protocol to understand changes in cardiac angiogenic pathways. A. A 3 mm incision is created on the dorsum of the pinna, and a subcutaneous pocket is created 0-24 h after injection of molecular, protein, or cellular treatments. B. An explanted neonatal (~24 h) heart is placed into the pocket and excess air is expressed from the pocket to facilitate adherence. C. Cardiac allografts are examined 7d post transplantation for visual viability and electrical activity.

et al., 2002) while controlling the age of the myocardium. Indeed, in the transplants, allograft neovascularization is mediated by host endothelial cells recruited into the donor hearts that recapitulate the cardiac myocyte-endothelial cell communication in vivo (Edelberg et al., 2002b; Aird et al., 1997), thus allowing the direct comparison of cardiac angiogenic potential in different age groups. Moreover, this model supports the testing of both molecular and cell-based interventions to modulate or restore functional cardiac angiogenesis in vivo. Indeed, the result of these studies are highly predictive of the functional role of different strategies in more clinically relevant models, including myocardial infarction studies (see Table 78.1), and thus provides a reasonable throughput screening approach in the investigation of cardiac angiogenesis in the aging rodent.

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