Biogerontology DOI 10.

1007/s10522-011-9325-8

RESEARCH ARTICLE

The developmental aging and origins of health and disease hypotheses explained by different protein networks
Bruno Cesar Feltes Joice de Faria Poloni Diego Bonatto

Received: 14 December 2010 / Accepted: 23 February 2011 Springer Science+Business Media B.V. 2011

Abstract One theory that attempts to explain how and why an organism ages is the developmental hypothesis of aging (DevAge), which describes how developmental programming leads to aging in adults. Interestingly, the developmental origins of health and disease hypothesis (DOHaD) asserts that some aging-associated diseases that occur in adults are closely related to development and to conditions in the intrauterine environment. Thus, both aging and agingassociated diseases can be viewed, at least in part, as the result of a developmental program that is activated early in embryogenesis and persists throughout the lifespan of the organism. We would expect this developmental program to be regulated by a set of interacting protein networks that connect environmental and

Electronic supplementary material The online version of this article (doi:10.1007/s10522-011-9325-8) contains supplementary material, which is available to authorized users.
B. C. Feltes J. de Faria Poloni Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS, Brazil D. Bonatto (&) Centro de Biotecnologia da Universidade Federal do Rio Grande do SulSala 219, Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do SulUFRGS, Avenida Bento Goncalves 9500Predio 43421, Caixa Postal 15005, Porto Alegre, RS 91509-900, Brazil e-mail: diegobonatto@gmail.com

molecular signals. However, the connection between aging and development is not clear. Thus, a systems biology approach that incorporates different omic databases for two mammalian models, Homo sapiens and Mus musculus, was used to evaluate how development and aging are interconnected. Interestingly, three major, evolutionarily conserved processes, namely the immune system, epigenetics, and aerobic metabolism, appear to regulate aging and development in both H. sapiens and M. musculus. Considering that these three processes are essential to embryogenesis, the protein networks within these processes are subjected to strong selective pressure to eliminate gross developmental abnormalities in early embryogenesis. This selective pressure becomes more relaxed in the adult organism, permitting the onset of aging-associated diseases and inammation-related aging; this concept echoes the antagonistic pleiotropy hypothesis of aging. Keywords Developmental aging (DevAge) Developmental origins of health and disease (DOHaD) Antagonistic pleiotropy hypothesis of aging Systems biology Inammaging

Introduction The mechanism of aging, which can be dened as the loss of physiological aptitude after an organism reaches its maximum reproductive capability

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(Vijg and Suh 2005), occurs in almost every living organism from prokaryotes to eukaryotes. Many hypotheses have been developed and tested to explain the aging process, and the resulting models attempt to explain aging at the organismal, organ, cellular, and molecular levels (Yin and Chen 2005). From those studies, other important hypotheses regarding the possible triggers of the aging process were dened, such as those concerning the accumulation of DNA mutations, free radical overproduction and accumulation, continuous cellular oxidative stress, and telomere shortening (Yin and Chen 2005). Despite intensive research, the mechanisms for how, when and why aging is initiated in multicellular organisms are virtually unknown. The antagonistic pleiotropy hypothesis (APH) of aging attempts to connect the evolutionary and molecular aspects of aging (Williams 1957; Leroi et al. 2005; Ungewitter and Scrable 2009). The APH denes aging as a consequence of the declining force of natural selection; traits that benet young organisms can have unspecied deleterious effects that lead to aging phenotypes later in the adult organism (Williams 1957; Leroi et al. 2005; Ungewitter and Scrable 2009). If these traits are activated early in life, e.g., during embryogenesis, and favor correct tissue development and maturation, then these mechanisms can escape natural selection but lead to aging later. Another unifying theory, the developmental theory of aging (DevAge), is garnering attention in the elds of both development and aging. DevAge, in its essence, was rstly proposed by the pioneering work of Dilman (1971, 1994) and further extended by the proteomic analysis performed by de Magalhaes and Church (2005). Briey, DevAge suggests that the aging process is the result of a developmental program that remains active for the entire lifespan of an organism (de Magalhaes and Church 2005), and this theory agrees with the basic requirements of APH. Such developmental programming will impact not only aging, but also other physiological processes. This idea is corroborated by the observation that tissue differentiation and body pattern formation in a fetus are mainly regulated by maternal nutrition and placental functions that maintain high rates of proliferation, growth and cell differentiation (Gluckman et al. 2010). Decient

supplies of macro- and micro-nutrients, oxygen and endocrine signals will impact not only the growth of the fetus, but also its future health (Gluckman et al. 2010). In fact, it was demonstrated that mineral deciencies during perinatal development can result in behavioral, immunological and biochemical abnormalities that persist into adulthood (Keen et al. 2003). Although these persistent defects can be partially attributed to subtle morphological abnormalities, they may be secondary to development-associated changes in DNA methylation patterns (Keen et al. 2003). Epigenetic defects combined with subtle morphological abnormalities can inuence an individuals risk for certain chronic diseases and thus inuence the risk for morbidity and mortality later in life (Keen et al. 2003). The origin of diseases and onset of aging processes was rstly explained by Thrifty Phenotype (THP) hypothesis (Hales and Barker 1992, 2001) and further expanded to the so-called developmental origins of health and disease (DOHaD) or developmental programming hypothesis (Langley-Evans 2006; Gluckman et al. 2010). In this scenario, an ensemble of molecular mechanisms senses the environment where the embryo is developing and translates these cues into biochemical information that will program both development and aging. An example of a molecular mechanism that could link DevAge and DOHaD is epigenetics. In embryonic stem cells (ESCs), epigenetic regulation is an essential factor for the differentiation of tissues and the formation of body patterns (Ringrose and Paro 2004; Bibikova et al. 2008). Additionally, many diseases that develop in adult organisms (e.g., type 2 diabetes or neurodegenerative diseases) are affected by epigenetic programming that occurs during embryonic development (Aagaard-Tillery et al. 2008). However, other molecular mechanisms that require further explanation are operating during embryonic development to program the fetus for adult life. Here, we examine the DevAge theory compared to the DOHaD hypotheses at the molecular level using a systems biology approach for two mammalian models: Homo sapiens and Mus musculus. Different physical proteinprotein interaction (PPPI) networks derived from mammalian interactome projects are described. Moreover, clustering analyses of the major biological processes derived from the PPPI networks

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and major centrality identication were conducted to evaluate the molecular and cellular mechanisms underlying the DevAge and DOHaD processes in H. sapiens and M. musculus.

Network centralities and local topological analyses Major network centralities (node degree and betweenness) were computed from the networks and local topologies. Bottleneck nodes were dened considering all nodes with the highest threshold generated by each centrality calculated by Centiscape 1.0 (Scardoni et al. 2009a, b). A PPPI subnetwork for H. sapiens and M. musculus containing the major bottleneck nodes was drawn from Centiscape 1.0 data. The number of shared bottleneck nodes between H. sapiens and M. musculus PPPI subnetworks was obtained by Venn analysis using the Area-Proportional Venn Diagram Plotter and Editor software [http://bioinforx.com/free/bxarrays/venndiagram.php]. Gene ontology analysis The protein motifs generated from MCODE were further studied by focusing on the major biologicalassociated processes using Biological Network Gene Ontology (BiNGO) software (Maere et al. 2005), which is a Cytoscape plug-in available at http://www. cytoscape.org. The degree of functional enrichment for a given cluster and category was quantitatively computed (P value) using hypergeometric distribution, and multiple test correction was also assessed by applying the false discovery rate (FDR) algorithm (Benjamini and Hochberg 1995), which was fully implemented through BiNGO software with a signicance level of P \ 0.05.

Materials and methods Interactome network design and global topological analysis To design binary physical proteinprotein interaction (PPPI) networks related to aging and development mechanism, the metasearch engine STRING 8.2 [http://string.embl.de/] was initially applied by using proteins classically described in literature as being related to development (e.g., HOX, PAX, and BMPs proteins); aging and epigenetics (e.g., histone deacetylases and sirtuins). The STRING 8.2 software allowed us to visualize the physical connection among proteins related to development, aging, and epigenetic processes. In this sense, the following parameters were used within STRING 8.2: active prediction methods all enabled except text mining; no more than 50 interactions; high condence score (0.700); and network depth equal to 2. In addition to STRING 8.2, the GeneCards [http://www.genecards. org/], PubMed [http://www.ncbi.nlm.nih.gov/pubmed/], Human Aging Genomic Resources (GenAge) [http:// genomics.senescence.info/genes/human.html], NetAge database [http://netage-project.org], and iHop [http:// www.ihop-net.org/UniPub/iHOP/] search engines were also employed using the default parameters. The results gathered from these search engines were subsequently analyzed using Cytoscape 2.6.3 (Shannon et al. 2003). To select the most relevant protein subnetworks from the interactome, the initial PPPI network was analyzed using Molecular Complex Detection (MCODE) (Bader and Hogue 2003), which is a Cytoscape plug-in freely available at http://baderlab.org/Software/MCODE. The parameters used in MCODE to generate the subnetworks were: loops included; degree cutoff 2; deletion of single connected nodes from cluster (haircut option enabled); expansion of cluster by one neighbor shell allowed (uff option enabled); node density cutoff 0.1; node score cutoff 0.2; kcore 2; and maximum depth of network 100. A MCODE score was calculated for each protein/small compound present in the interactome networks.

Results Analyses of interactome networks and subnetworks associated to embryonic development, aging, and epigenetics in H. sapiens and M. musculus The increasing number of available omic databases has enabled the development of new systematic tools for understanding the interplay between regulatory processes found within a cell. Considering a potential connection between DevAge and DOHaD, we developed a systems biology study by investigating two binary PPPI networks and their associated pathways

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using the data available for H. sapiens and M. musculus (Fig. 1). In this sense, a PPPI network that could link well described proteins in the literature (whose functions have been observed in adult tissues) to the aging and development processes (Fig. 1) was searched for both organisms. These networks were expected to contain

Fig. 1 Experimental approach employed to retrieve agingand development-associated interactome data from human and mouse proteinprotein interaction databases. The interactome data were obtained from an initial query using aging- and development-associated proteins, resulting in two physical proteinprotein interaction (PPPI) networks. These two networks were analyzed by Cytoscape software, and the union function of the Merge networks plugin was used to fuse both networks into a unique PPPI network. This nal network was then analyzed by MCODE to identify the major associated subgraphs and by Centiscape to retrieve important nodes by centralities analysis. Biological processes found within subgraphs were retrieved by employing the BiNGO plugin of the Cytoscape software. The information retrieved from literature data mining and network analyses was employed to design a model of how development interplays with aging in human and murine embryos and adult organisms (for more details, please refer to Supplementary Material 1)

proteins that participate in aging and development and have already been described in the literature, such as PAX, HOX, DNA methyltransferases (DNMTs), sirtuins, and histone deacetylases, among others. The data gathered demonstrated that the topologies of PPPI networks are very similar for both H. sapiens (Fig. 2) and M. musculus (Fig. 3). The PPPI network of H. sapiens (Fig. 2) contained 292 nodes and 2,559 connectors, while the PPPI network of M. musculus (Fig. 3) contained 331 nodes and 2,301 connectors. Both PPPI networks were composed of three subnetworks (Figs. 2, 3), each representing different biological processes (Supplementary Material 1). For H. sapiens-associated PPPI network (Fig. 2), the three subnetworks can be classied into: (i) inammatory response/immune system process; (ii) regulation of transcription/chromatin remodeling/glucose metabolism; and (iii) multicellular organismal development. For the PPPI network of M. musculus (Fig. 3), the three subnetworks are: (i) immune system/inammatory response; (ii) regulation of transcription/development; and (iii) aerobic respiration/glycolysis. Additionally, the networks gathered for H. sapiens (Fig. 2) and M. musculus (Fig. 3) and their corresponding subnetworks (Supplementary Material 2) contained proteins that participate in biological processes such as the inammatory response, transcription regulation, epigenetics, glucose metabolism, and developmental processes (Supplementary Material 1). Interestingly, a comparison of PPPI network of H. sapiens (Fig. 1) with the common signature of longevity and age-related disease proteins (ARDs) network (containing 643 nodes), as described in NetAge database, indicated that 57 nodes are commonly present in both networks and distributed within the subnetworks (Supplementary Material 1). The topological similarities between H. sapiens (Fig. 2) and M. musculus (Fig. 3) networks prompted us to ask if common pathways or mechanisms of aging and development should be found in these two biological models. By applying centrality analysis to PPPI networks of H. sapiens and M. musculus, evolutionarily conserved pathways and nodes that represent a potential mechanism for the molecular control of DevAge/DOHaD processes were identied. Thus, for both the H. sapiens (Fig. 2) and M. musculus (Fig. 3) networks, we obtained two small subnetworks containing only bottleneck nodes (Fig. 4a and b, respectively). A third subnetwork,

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Fig. 2 A physical proteinprotein interacting (PPPI) network of major proteins described in the literature as associated with aging and development in H. sapiens. Both clustered proteins

(composing different subnetworks) and unclustered proteins are represented by nodes of different shapes and colors, as indicated in the inset

containing common (or shared) bottlenecks that dene evolutionarily conserved points of control was further analyzed (Fig. 5). Identication of important nodes within DevAge/ DOHaD-associated interactome networks and subnetworks by centrality analyses Two major centrality analyses, node degree and betweenness, were performed in this work to identify the most important nodes within the PPPI networks of H. sapiens and M. musculus. Node degree is the simplest centrality measure in a given network, and it corresponds to the number of nodes adjacent (directly connected) to a given node (Scardoni et al. 2009a, b). The node degree represents the popularity of a given node, and highly connected nodes in a network are termed hubs (Yu et al. 2007). Betweenness indicates the extent to which a specic node is

between all other nodes within the network (Newman 2005). In general, betweenness shows the inuence of a node over the spread of information throughout the network. The measure of node degree and betweenness gives rise to a third piece of information called node bottleneck, which is dened as all nodes with high betweenness values. These nodes are central points that control the communication between other nodes within the network. Bottleneck also indicates all nodes that are between highly interconnected subgraph clusters, and removing a bottleneck could divide a network (Girvan and Newman 2002; Yu et al. 2007). Thus, for the human subnetwork, there were 69 bottleneck nodes (Fig. 4a; Supplementary Material 3), while the murine bottleneck-associated subnetwork was formed by 60 nodes (Fig. 4b; Supplementary Material 3). Moreover, a Venn diagram analysis of the human and murine bottleneck-associated

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Fig. 3 A PPPI network of major proteins associated with aging and development in M. musculus. Clustered proteins (composing different subnetworks) and unclustered proteins are represented by nodes of different shapes and colors, as indicated in the inset Fig. 4 Smaller PPPI subnetworks derived from calculated centrality analyses for H. sapiens (a) and M. musculus (b). The color and shape of each node indicates the associated subnetwork (subnetworks 13 and unclustered proteins; please refer to the main text of the manuscript for more details)

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subnetworks identied 40 shared proteins, which represent approximately 58 and 66% of the total nodes observed in the human and murine bottleneckassociated subnetworks, respectively (Fig. 5; Supplementary Material 3).

Discussion Interactome networks to link proteins associated with embryonic development, aging, and epigenetics in H. sapiens and M. musculus The inammation-epigenetic subnetwork and fetal intrauterine growth restriction linked to aging and epigenetic mechanisms From the different subnetworks observed within the major interactome networks of H. sapiens (Fig. 2) and M. musculus (Fig. 3), the inammation-epigenetic cluster contains very interesting proteins that could potentially connect DevAge and DOHaD. It has been reported that inammation was implicated in embryo development by the participation of macrophages during the initial phase of mammalian development (Ovchinnikov 2008). In this sense, the inammatory subnetworks for both H. sapiens and M. musculus contain cytokines and proteins that stimulate macrophage activation, such as interleukin 18 (IL18) and toll-like receptor 4 (TLR4; Supplementary Material 2). Macrophages present in embryonic tissues express a large number of cytokines and mitogenic factors, including IGF1, TGFa, PDGFA, TGFb, ILs, VEGFs, RANKL and LIF (Rappolee and Werb 1988; Hume et al. 2002; Ovchinnikov 2008), which probably stimulate cell proliferation and differentiation. Interestingly, a pro-inammatory cascade observed at the feto-maternal interface (Hess et al. 2007) culminates in NF-jB nuclear translocation and production of secondary mediators, such as chemokines, COX-2, prostaglandins, MMPs, and pentraxin 3 (PTX3), and to the expression of adhesion molecules (Dinarello 1996; Medzhitov 2001; ONeill 2002; Garlanda et al. 2008). Once the feto-maternal interface is established by inammatory cytokines, the coordinated expression of angiogenic growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor, and their respective receptors on invasive trophoblasts occurs (Leonard et al. 2006). Impaired endovascular trophoblast invasion causes inadequate conversion of the uterine arteries and reduced uteroplacental blood ow, leading to fetal intrauterine growth restriction (IUGR) (Redman and Sargent 2005; Garlanda et al. 2008). Fetal IUGR has been associated with premature aging and the

Fig. 5 Area-proportional Venn diagram of the H. sapiens (a) and M. musculus (b) centrality-associated subnetworks. The number of total proteins, the degree of interpolation between subnetworks and the number of unique proteins for each subnetwork are indicated in the Venn diagram. A small PPPI subnetwork derived from proteins that are commonly found in both H. sapiens and M. musculus centrality-associated subnetworks is shown in the box

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development of aging-associated diseases of adult individuals in various mammalian models (Jennings et al. 1999; Chen et al. 2009; Warner and Ozanne 2010). The connection between the inammation/immune systems (cluster 1; Figs. 2, 3) and transcriptional regulation/chromatin remodeling (cluster 2; Figs. 2, 3) is a poorly understood aspect of the DevAge/ DOHaD mechanisms. Some major proteins found in both the human and murine cluster 2 (Figs. 1, 2) were as follows: (i) DNA methyltransferase 1 (DNMT1), DNMT3a and DNMT3b; (ii) nuclear or class I histone deacetylase 1 (HDAC1), HDAC2, and HDAC3; (iii) class III HDACs termed sirtuin 1 (SIRT1), and SIRT2; and (iv) p300 (histone acetylase; HAT) (Supplementary Material 2). These proteins belong to major complexes that alter the state of chromatin in response to environmental and/or phys iological conditions (Backdahl et al. 2009), leading to gene silencing or gene expression induction (Sadoul et al. 2008). In addition to the connection observed between the immune system/inammatory process and the epigenetic control of gene expression during development, other important proteins were also observed in cluster 2 in both the H. sapiens and M. musculus networks (Figs. 2, 3). These proteins are the tumor suppressor protein p53 and transcription factors, such as the HOX and PAX proteins, that control body pattern formation and organogenesis. The role of intestine development, immune system, and glucose metabolism on aging The p53 protein is important not only for its role in cell survival, but also for its debated role as an aging or anti-aging protein (Zafon 2007). In fact, our data analysis indicated that p53 is connected with CDX2, which is linked to the inammatory/immune subnetwork (Fig. 1). CDX2 is a homeobox protein associated with early differentiation and maintenance of the intestinal epithelium in both the small and large intestines (Suh and Traber 1996). Because the promoter region of CDX2 contains an NF-jB binding site, it is clear that immune and inammatory processes at early embryogenesis can modulate the expression of this gene and affect intestinal development. The intestines of IUGR neonates have reduced weight (proportionate to body weight), length, wall thickness,

villous height, and crypt depth (Baserga et al. 2004). A consequence of these intestinal changes induced by IUGR is that the neonate immune system becomes compromised because the intestinal barrier is involved in the rst steps of postnatal immune system maturation, protection against food allergens, and protection against environmental microorganisms, affecting aging at later life (Schumann et al. 2005; Willing and Van Kessel 2009). Notably, lymphocytes rst appear in the intestinal subepithelium early in development, indicating a strong connection between the digestive and immune systems during development (VeeremanWauters 1996). Another interesting occurrence observed in both PPPI networks was the presence of a subnetwork associated with glucose metabolism and aerobic respiration (Figs. 2, 3). The presence of citric acid cycle enzymes such as succinate dehydrogenase complex members (SDHA and SDHB; Figs. 2, 3; Supplementary Material 1), aconitase (ACO1 and ACO2; Figs. 2, 3; Supplementary Material 1), and enzymes associated with glucose metabolism such as glycerol-3-phosphate dehydrogenase 1 and 2 (GPD1/ GPD2; Figs. 2, 3; Supplementary Material 1) indicated the importance of these metabolic pathways during embryonic development. Other peptides/proteins that are important for glucose homeostasis and metabolism are also present in the PPPI networks and include insulin/insulin growth factor 1 (IGF-1) and mTOR (Olovnikov et al. 2009). The data gathered from the systems biology analysis showed that IGF-1 is a node that is connected to SIRT1 and p53 (Figs. 2, 3; Supplementary Material 2). Interestingly, it was reported that lower baseline IGF-1 levels during embryogenesis predict the subsequent development of impaired glucose tolerance, type 2 diabetes and cardiovascular disease, all chronic conditions associated with aging in adults (Chakravarthy et al. 2008). The role of molecular oxygen homeostasis on development and aging In addition to their roles in embryonic glucose and general metabolism, both IGF-1 and mTOR are linked to hypoxia-inducible factor-1, subunit a (HIF1A; Figs. 2, 3), which is a transcription factor essential for cellular and systemic homeostatic responses to hypoxia.

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During early embryogenesis, HIF1A is activated in response to inammation and low O2 tension (Imtiyaz and Simon 2010). This early embryogenesis environment induces the differentiation of various stem cell progenitors, including the differentiation of early mesoderm into hemangioblasts (Fraisl et al. 2009), which results in the early steps of blood vessel development through paracrine release of angiogenic cytokines, such as VEGF, by hematopoietic cells. Our data support the role of HIF1A in embryogenesis because this protein is directly linked to HDACs, VEGF nodes, and other proteins related to development and aging (Figs. 2, 3; Supplementary Material 2). Molecular oxygen can also modulate the epigenetic process by regulating HDACs (Okazaki and Maltepe 2006). During early embryogenesis, hypoxia and HIF1A expression correlate with HDAC1 and VEGF activation and result in angiogenesis (Okazaki and Maltepe 2006). Nonetheless, the role of O2 in DOHaD is not clear, despite the observation that mouse embryos cultured in 20% oxygen in vitro before reimplantation show greater perturbations in global epigenetic patterns than those cultured in 5% oxygen or those that developed in vivo (Rinaudo et al. 2006). In humans, such perturbations cause lessthan-optimal embryonic development (Waldenstrom et al. 2009) and lower rates of implantation and live births (Waldenstrom et al. 2009) after culture in an atmospheric (1921%) oxygen environment. Interestingly, a condition termed placental hyperoxia has been described, in which high oxygen levels in the intervillous space of the placenta downregulate VEGF, restrict angiogenesis and lead to IUGR (Khaliq et al. 1999).

A common regulatory pathway for development and aging in H. sapiens and M. musculus The bottleneck nodes shared by humans and mice contained important aging- and development-associated proteins (Fig. 5; Supplementary Material 3), such as p53, EP300, HOXs, HDAC1, SIRT1, and NFjB. It is important to note that a similar work published by de Magalhaes and Toussaint (2009) reports a connection between development and aging by exploring an H. sapiens proteomic network, supporting our data.

Moreover, other interesting aging- and development-associated bottleneck nodes were also represented in this shared bottleneck subnetwork, including PAXs, BMP4, RAD50, and SMAD4 (Fig. 5; Supplementary Material 3). The functions of the PAX proteins are well known in development and stem cell maintenance (Lang et al. 2007). The PAX proteins are transcription factors that, together with HOXs, coordinate organogenesis (Plaza et al. 2008). In this work, three PAX proteins are described: PAX2, PAX6, and PAX9 (Fig. 5; Supplementary Material 3). PAX2 is necessary for the development of the urogenital tract, eyes, and CNS, while PAX6 acts in the formation of the eyes, nose, central nervous system and pancreas. PAX9 is required for the development of the thymus, parathyroid glands, ultimobranchial bodies, teeth, skeletal elements of the skull, larynx, and distal limbs (http://www.genecards.org). Interestingly, it has been demonstrated that IUGR induced by maternal protein restriction in a murine model leads to hypertension and a reduced number of glomeruli in the kidneys, which affects lifespan, especially when these animals are exposed to a high-protein diet (Chen et al. 2010). In addition, IUGR induces atrophy of the thymus (Cox and Marton 2009) and affects the development of pancreatic islets by altering the epigenetic mechanisms of these cells, leading to the development of type 2 diabetes (Thompson et al. 2010). These facts show that PAX proteins appear to be important targets of epigenetic control, and our data support this hypothesis in the context of major bottleneck proteins (Fig. 5). Unfortunately, and despite the intensive research related to PAXs, it is not completely clear how these proteins acts in aging. BMP4 is another interesting bottleneck that appears in the subnetwork (Fig. 5; Supplementary Material 3). This protein is necessary for cartilage and bone formation through mesoderm induction, tooth development, limb formation and fracture repair (http://www.genecards.org). While the role of BMP4 in aging is less understood, some reports have indicated that BMP4 is an important mediator of atrophic age-related macular degeneration because it mediates oxidative stress-induced retinal pigment epithelium senescence in vitro via the SMAD and p38 pathways (Zhu et al. 2009). In addition, overexpression of BMP4 was able to induce premature senescence in lung cancer cells (Su et al. 2009). The role of

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BMP4 in epigenetic regulation and in IUGR is also not well understood, but a recent article by Murohashi et al. (2010) showed that trophoblast cells produce BMP4 by means of CDX2 to promote the proper growth of early mouse embryos. Because early embryogenesis correlates with rapid mitosis of ESCs (Luo et al. 1999), it is not surprising that DNA damage, especially DNA single- and double-strand breaks (SSBs and DSBs, respectively), could accumulate during this period. In fact, research has shown that DNA repair proteins are implicated in early embryogenesis (Chuykin et al. 2008). Similarly, RAD50 (Fig. 5; Supplementary Material 3), which is a major component of the RAD50/MRE11/NBS1 (MRS) complex that acts in DNA repair and chromatin remodeling (Iijima et al. 2008), was present in the bottleneck-associated subnetwork. RAD50 is important during embryogenesis, and RAD50 mutant ESCs in a murine model are not viable, especially during the phase of accelerated cell proliferation (Luo et al. 1999). The failure of cells to proliferate could be attributed to the accumulation of unrepaired or misrepaired spontaneous DSBs over several cell cycles (Luo et al. 1999). Normally, however, rapid cell cycling and the formation of DSBs could be the initial trigger that results in the secretion of IL-6 and/ or other inammatory cytokines, which promote angiogenesis and cell differentiation, and that has been observed for some tumor types (Rodier et al. 2009). Finally, SMAD4, a bottleneck node that is highly connected with PAX, BMP, p53 and other proteins (Fig. 5; Supplementary Material 3), was conserved between murine and human models. The role of SMAD4 in embryogenesis has been well documented (Costello et al. 2009), and SMAD4-null embryos arrest shortly after implantation due to defects in the extra-embryonic lineages (Yang et al. 1998; Chu et al. 2004). Additionally, SMAD4 has important roles in inammation and hematopoiesis (Karlsson et al. 2007) because this protein is necessary for promotion of hematopoietic stem cell renewal, as well as maintenance of colon structure and homeostasis (Karlsson et al. 2007). Other nodes could be observed in the bottleneckassociated subnetwork, including the SWI/SNFrelated, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4; Fig. 5 and Supplementary Material 3); the nuclear

receptor coactivator 3 (NCOA3; Fig. 4 and Supplementary Material 3); the transcription factor YY1 (Fig. 5 and Supplementary Material 3); and the polycomb group RING nger protein 4 (BMI1; Fig. 5 and Supplementary Material 3). These proteins were described as necessary for some aspects of development and for the control of epigenetic mechanisms (Kim et al. 2006; Magnani and Cabot 2009; Kashyap and Gudas 2010), but their impact on aging is less understood, making these proteins interesting targets for DevAge/DOHaD studies.

A model linking DevAge with DOHaD in an antagonistic pleiotropy view The idea that the developmental changes that occur in the intrauterine environment can have impacts on adult organisms can be found initially in the Thrifty Phenotype (THP) hypothesis. The THP hypothesis posits that during evolution a set of key genes (thrifty genes) were selected to provide resistance to starvation by triggering a fast insulin response that would facilitate the restoration of adipose tissue (Hales and Barker 1992, 2001). The hypothesis also suggests that this adaptive response in pregnant individuals could cause the regulation and growth of critical organs in the fetus while being simultaneously capable of causing the malformation of other organs, leading to changes in fetal and adult metabolism (McMillen and Robinson 2005). Moreover, the idea that the embryonic process can adaptively respond to specic intrauterine changes raises the hypothesis that these errors in the developmental process could program certain conditions later in life because these early settings could predetermine metabolic function and morphological conditions (McMillen and Robinson 2005). Thus, essential responses or a process that takes place during embryonic development could be detrimental later in life. The THP hypothesis was further expanded to the so-called Predictive Adaptive Response (PAR) hypothesis (Gluckman and Hanson 2006), which proposed that the fetus dynamically interacts with and assesses the environment into which it will be born and adapts to gain a future survival advantage (Gluckman and Hanson 2006). Finally, both the THP and PAR hypotheses were joined in DOHaD, which considers extrinsic and intrinsic factors that affect the developmental

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program by changing the expression of specic genes associated with development and epigenetic control (Jones and Ozanne 2009). The accepted denitions of DOHaD and, as described before, of DevAge can be better understood if we consider the mechanism of APH. Both DOHaD and DevAge presuppose that early embryogenesis and its molecular pathways of development, which are regulated by the intrauterine environment, dene the lifespan of the organism, including aging and aging-associated diseases. Thus, the molecular pathways involved in development and differentiation are strongly subjected to natural selection and other evolutionary pressures (Richardson 1999). Epigenetic alterations (as a consequence of the embryo-associated environment) affect gene expression in developing tissues in response to selection to act against individuals that show gross anatomical or physiological abnormalities (Richardson 1999). Our model includes DOHaD/DevAge in the same conditions of development (Fig. 6), in which initial embryogenesis occurs in a low O2 environment, leading to vasculogenesis and early oxidative stress signaling that culminate with the production of proinammatory cytokines by the mother and by the differentiating ESCs (Fig. 6). Once the vascular connection occurs between the placenta and fetus, there is a gradual increase in the fetal tissue O2 concentration, which establishes aerobic respiration in the fetus and starts the developmental program. Under normal conditions, adequate levels of O2 and aerobic metabolism activate the developmental/ epigenetic program by regulating a series of proteins that target the chromatin structure, controlling the progression of the cell cycle and cell fate. During this phase of embryogenesis, fetal stem cell niches are formed in various tissues (Fig. 6), including intestinal, skin, neural, germ line, and hematopoietic tissues (Li and Xie 2005). These niches have important roles in the maintenance of tissue homeostasis during the adult life because they are required for functions such as wound healing and immune system modulation (Li and Xie 2005). The three major processes observed in our systems biology data model (immune system/inammation, development/epigenetics, and aerobic respiration/ glucose metabolism; Fig. 6) possibly trigger the aging mechanism at the cellular and molecular levels because aging itself is important for tissue

differentiation and maturation. These processes should be strongly subjected to selective pressure (Akam 1998; Richardson 1999), and the same is most likely true for embryo-associated aging. This evolutive surveillance mechanism ensures that the embryo will develop correctly. When the fetus is establishing the placental connection with the mother, the intrauterine environment is strongly regulated by external factors, such as the mothers nutritional status (Fig. 6). These environmental factors can induce epigenetic effects during early development to inuence various stable gene expression patterns that persist even after the disappearance of the initial signal and may continue to determine a distinct phenotype (Vaiserman 2008). In the adult organism, the same three major processes that are directly associated with DevAge/ DOHaD (Fig. 6) also participate in homeostasis. For example, wound healing is an essential mechanism that maintains the viability of adult tissues. The response to wounding is composed of three welldescribed and interdependent phases: (i) inammation, (ii) new tissue formation, and (iii) tissue remodeling (Gurtner et al. 2008). The initial reaction following cutaneous wounding is the production of inammatory mediators, including IL-1, TNFa, and IL-6, in the dermis and epidermis. This rst reaction is important for the initial removal of cellular debris and for the regulation of sequential responses after wounding, such as tissue formation and remodeling (Adams 2009). Overall, the processes of wound healing and immune modulation require the intense participation of adult stem cells (Rando 2006), which are mobilized from their niches (Rando 2006; Fig. 6). Glucose metabolism and aerobic respiration also have important roles in the aging and development of an adult organism. Chronic diseases, typically associated with aging and mainly regulated by environmental factors, e.g., type 2 diabetes (Kawahito et al. 2009), can affect the metabolism of glucose by inducing so-called glucose toxicity, a clinical condition in which hyperglycemia itself reduces the insulin secretion capacity of pancreatic b-cells, and the resultant increase in insulin resistance leads to further hyperglycemia. This vicious cycle nally leads to the inability of b-cells to secrete insulin (Kawahito et al. 2009). As described before, insulin resistance could be pre-determined during embryogenesis under IUGR conditions.

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Fig. 6 Aging and development as unique processes in H. sapiens and M. musculus as observed by systems biology analysis. Embryogenesis takes place in the intrauterine environment, where the placenta connects the developing embryo with the mothers major physiological systems, such as nutrient acquisition and metabolism. In addition, the placenta translates signals from the mothers environment to the embryo. The establishment of the embryoplacentamother interface occurs together with the activation of the immune system/inammatory process, epigenetics/development process, and glucose metabolism/aerobic respiration in embryonic tissues during early embryogenesis. Initially, the secretion of pro-inammatory cytokines by the mothers immune system due to the placentaembryo connection, some embryonic structures (e.g., the intestine), and the low O2 tension observed in the uterus stimulate embryo tissue differentiation. As a consequence of this developmental process, a molecularcellular aging mechanism is established in the embryo that facilitates differentiation. Additionally, during embryogenesis, stem cell niches are formed in different tissues. The connection of the mother to the embryo by the placenta determines how the embryo will develop and age (developmental aging; DevAge) by adapting the embryo to the intrauterine and

maternal environmental conditions. This environmental adaptation, which is mainly regulated by the immune/epigenetics/ aerobic metabolism processes, can also predispose the organism to chronic diseases or physiological dysfunctions (developmental origins of health and disease; DOHaD). It should be noted that the fetal immune system, epigenetics and development mechanisms, and glucose/aerobic metabolism are strongly subjected to natural selection, eliminating poorly adapted embryos. However, all surviving embryos that adapt to the uterine environment have the potential to develop into an adult individual. The same mechanisms that act during embryogenesis are also observed in adult tissues, promoting the physiological homeostasis of the organism and mobilizing adult stem cells from their niches to repair damaged tissues. However, unlike during embryonic development, the selective pressure becomes relaxed as the organism ages, allowing the accumulation of damage (e.g., oxidative damage) to molecules, biological processes, and tissues. Relaxed selective pressure allows the development of aging-associated chronic diseases (e.g., type 2 diabetes and cardiovascular diseases) as well as a mildly systemic chronic inammation that correlates with aging in adults

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Hyperglycemia, in turn, increases the production of ROS, leading to the formation of advanced glycation end products (AGEs) in tissues (Stoppera et al. 2003; Baynes 2001). A large body of evidence suggests that AGEs are important pathogenic mediators for various diseases, such as diabetic complications, Alzheimers disease, atherosclerosis, and dialysis-related amyloidosis (Hou et al. 1997; Yan et al. 2003; Sun et al. 1998). Additionally, AGEs can induce inammation in different tissues by binding to the AGE receptor (AGER) (Bierhaus et al. 2005). Interestingly, the activation of AGER by AGEs released from cell debris can contribute to the wounding response in a non-aged organism by inducing acute inammation, which is an essential condition for tissue healing (Adams 2009). However, it is also possible that chronic activation of the wound response pathway by AGE accumulation and AGER signaling due to impaired glucose metabolism could increase the rate of tissue senescence by activating inammation. Furthermore, senescence has been demonstrated to facilitate wound healing under normal physiological conditions (Adams 2009), supporting our idea that aging is a fundamental process for tissue differentiation and maturation. However, the overstimulation of wound healing may ultimately become a source of tissue damage and may even cause age-associated tissue degeneration, cancer (Adams 2009), or inammaging (Fig. 6). Considering APH, the strong selective pressure observed during embryogenesis becomes relaxed as the adult organism ages. The cumulative damages induced by glucose metabolism/aerobic respiration, epigenetic modulation/development process, and immune system/inammation augment the predetermined conditions that originated from the DevAge/DOHaD programming, leading to agingassociated chronic diseases (Fig. 6). It should be noted that the relationship between aging-associated chronic diseases and development has been recently described by means of network analyses (Budovsky et al. 2009). In this sense, a comparison of the common signature of longevity and age-related disease proteins (ARDs) network present in the NetAge database (Tacutu et al. 2010) and described in the manuscript of Budovsky et al. (2009), with the PPPI network of H. sapiens, indicated the presence of 57 common proteins, all participating in epigenetic mechanism, development, and aging.

Interestingly, it has been proposed that epigenetic regulation could have a so deep impact in aging and aging-associated chronic diseases that the term epigenetic antagonistic pleiotropy (EPH) was proposed, similarly to APH (Budovsky et al. 2006). The concept of EPH could be easily adapted to development, where a strong natural selection force molds the global epigenetic patterns of a developing embryo, and consequently denes how an organism will age and will develop aging-associated chronic diseases (Attig et al. 2010; Munoz-Najar and Sedivy 2011). Noteworthy, experimental data obtained from C. elegans using RNAi for the screening of longlived individuals showed that early gene inactivations of essential genes associated to development, metabolism, and chromatin structure caused growth arrest at larval stages, resulting in long-lived animals (Curran and Ruvkun 2007). In conclusion, the systems biology data gathered in this review indicate that the mechanisms that modulate early embryogenesis and aging in mammals are strongly regulated by a network of different proteins that connect both aging and development and are subjected to environmental regulation. In other words, intrauterine factors epigenetically regulate the adaptability of the embryo to its environment. Once viable, the organism will develop to adulthood. The same mechanisms that controlled early embryogenesis are still present in the adult but are less subject to natural selection pressures, which results in the origin of environment-associated chronic diseases and aging.
Acknowledgments This work was supported by research grants from the Conselho Nacional de Desenvolvimento Cientco e Tecnologico (CNPq; Grant number 471769/2007-0 and 300301/2009-0) and the Programa Institutos Nacionais de Ciencia e Tecnologia (INCT de Processos Redox em Biomedicina-Redoxoma; Grant number 573530/2008-4). Contract/grant sponsor: CNPq.

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