The roles of nitric oxide in murine cardiovascular development

Developmental Biology 292 (2006) 25 – 33 www.elsevier.com/locate/ydbio

Review

The roles of nitric oxide in murine cardiovascular development Anjali K. Nath a,b , Joseph A. Madri b,⁎ a

b

Yale University, Department of Molecular, Cellular and Developmental Biology, New Haven, CT 06520, USA Yale University School of Medicine, Department of Pathology, 115 Lauder Hall, 310 Cedar Street, New Haven, CT 06520, USA Received for publication 15 August 2005; revised 22 November 2005; accepted 16 December 2005 Available online 25 January 2006

Abstract Nitric oxide (NO) participates in a diverse array of biological functions in mammalian organ systems. Depending on the biochemical environment, the production of NO may result in cytoprotection or cytotoxicity. The paradoxical actions of NO arise from the complexities generated by the redox milieu, NO concentration/bioavailability, and tissue/cell context, which ultimately result in the wide range of regulatory roles observed. Additionally, in physiological versus pathological states, NO often displays diametrically opposing affects in several organ systems. Here, we will discuss the roles of NO during reproduction, organ system development, in particular, the cardiovascular system, and its potential implications in diabetes-induced fetal defects. © 2005 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Development; Cardiovascular; Diabetes; Vasculopathy; Vitelline; Yolk sac

Introduction The small polyvalent molecule, nitric oxide (NO), is generated by three conserved nitric oxide synthases (NOSs) isozymes: endothelial NOS (eNOS; Type III NOS (NOS-3)) located on human chromosome 7q35–7q36; inducible NOS (iNOS; Type II NOS (NOS-2) macNOS, hepNOS) located on human chromosome 12q24.2–7q36; and neuronal NOS (nNOS; Type I NOS (NOS-1) bNOS) located on human chromosome 17cen–17q11.2; and the recently discovered mitochondrial NOS (mtNOS) (Lacza et al., 2005; Moncada et al., 1997; Nicotera et al., 1999). The control of NOS isoform expression is complex, involving transcriptional and post-transcriptional mechanisms triggered by multiple signaling pathways during development, growth, homeostasis, and in a broad range of pathophysiological conditions (Kleinert et al., 2004; Li et al., 2002; Wang et al., 1999). In addition to the complex regulation of NOS expression, NOS isoform subcellular localization, association with receptors, and catalysis are modulated by a large diverse number of distinct binding proteins, some of which are capable of binding to e-, i-, and n-NOS (calmodulin), while others bind to individual isoforms (Dynamin-2 to eNOS, ⁎ Corresponding author. Fax: +1 203 785 7303. E-mail address: [email protected] (J.A. Madri). 0012-1606/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.12.039

or subsets of NOS isoforms (caveolin-1 to e- and i-NOS— albeit having distinct effects on the particular isoform) (Kone et al., 2003; Zhang et al., 2003). NOSs catalyze the oxidation of L-arginine to NO and Lcitrulline, using the cofactors, NADPH, FAD, and tetrahydrobiopterin (BH4). The constitutively expressed eNOS and nNOS additionally require Ca2+-calmodulin. Upon generation, NO freely diffuses through the cell membrane into the extracellular space reaching tissues up to a distance of 100 μm in a few seconds at 37°C, limited only by its half life and chemical reactivity. Subsequently, NO modifies protein thiols (S-nitrosylation or disulfide formation) or cysteine residues (to sulfenic and sulfinic acid). Additionally, NO can interact with free radicals to induce a variety of biological responses (Lacza et al., 2005; Nicotera et al., 1999; Stamler, 1994; Stamler et al., 1992). NO is a pleiotropic molecule that acts as a vasoactive agent, signaling molecule, neurotransmitter, and free radical in mammalian systems. NO participates in numerous biological functions including gene expression, cell growth, immune response, and matrix remodeling in several organ systems including the central nervous, cardiovascular, and immune systems. NO mediates these responses by interacting with several signaling pathways. The classic action of NO is activation of guanylate cyclase that subsequently acts on target proteins. Additionally,

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NO signals through several kinase cascades including mitogenactivated protein kinase (MAPK), janus kinase (JAK), and cJun N-terminal kinase (JNK) pathways. Transcription of several gene classes, such as cytokines, pro-inflammatory molecules, matrix proteins, matrix metalloproteinases, and hormones, is regulated by NO. NO also modulates reactive oxygen depending on signaling pathways by combining with reactive oxygen species (ROS) (Brune et al., 2001; Pineda-Molina and Lamas, 2001) which have been demonstrated to play a role in growth factor signaling including NGF- and VEGF-mediated signaling (Akassoglou, 2005; Erwin et al., 2005). NO plays a dual role as a protectant and antagonist in disease states (Chen et al., 2005; Martins et al., 1998; Moore and Hui, 2005; Nicotera et al., 1999). The biology of NO is quite complex but can be understood by examining its bioavailability, chemistry, and the redox microenvironment (Brune et al., 1999; Naseem, 2005; Nicotera et al., 1999). NO can directly react with molecules (metalloproteins) or can react with transition metals, oxygen, or superoxide anion (ROS) producing potent reactive nitrogen species which then oxidize or nitrosylate targets, some of which are antioxidants. NO binds two reactive oxidants produced by H2O2, metalloxo- and peroxospecies. Additionally, NO induces cyclooxygenase-2, a molecule found to protect against apoptosis in macrophages and cardiomyocytes by up-regulation of CuZn-superoxide dismutase, heme oxygenase-1, heat shock protein 70, Mn-superoxide dismutase, and Bcl-2 (Brune et al., 1999). NO and reactive nitrogen species are far less genotoxic than ROS. However, NO rapidly combines with ROS to produce a variety of highly reactive secondary species which depending on the concentrations of both ROS and NO create unique redox environments that are detrimental to cell systems in numerous disease states (Brune et al., 1999; Nicotera et al., 1999). NOS knockout mouse phenotypes While pharmacological approaches have provided significant insights into the dynamic and complex roles of NO in cellular, organ, and organismal biology, this approach is limited. The generation and use of single and multiple NOS isoform deficient mice have provided an independent approach to elucidate the distinct and shared roles of NOS isoforms (Gregg, 2003; Mashimo and Goyal, 1999). Single NOS knockout mice have aided investigators in resolving the complexities of the NO signaling systems, as well as the redundancy and compensation that occur in the absence of individual isoforms (Tranguch and Huet-Hudson, 2003). For example, retinal vascularization in the eNOS knockout is normal due to redistribution of nNOS to the deep capillary plexus leading to normal levels of NO in the endothelium (Al-Shabrawey et al., 2003). Similarly, compensation occurs by the generation of splice variants that lead to persistent low enzyme activity (b5% wildtype NO production), allowing development to proceed normally. Furthermore, these splice variants often display tissue specificity leading to unique affects on organ system development (Forstermann et al., 1998; Huber et al., 1998; Kolesnikov et al., 1997; Mashimo and Goyal, 1999; Panda et al.).

Interestingly, the generation of NOS isoform double knockout mice did not result in decreases in litter sizes (Tranguch and Huet-Hudson, 2003) but did result in significant decreases in the generation of double knockout pups (12 compared to the expected 48 out of 773 pups) (Tranguch and Huet-Hudson, 2003). These data are consistent with the concept that double NOS knockouts exhibit a developmental abnormality resulting in perinatal loss, although the stage at which the conceptuses are lost is currently unknown as are the specific developmental anomalies. Recently, a triple NOS knockout mouse was generated (Morishita et al., 2005). These mice exhibited marked reductions in survival and fertility rates and higher systolic blood pressure (similar to that observed in the single and double knockout mice). Additionally, the surviving mice were noted to exhibit hypotonic polyuria, polydipisia, and unresponsiveness to vasopressin, consistent with a picture of nephrogenic diabetes insipidus. The full impact of NOS dysregulation and/or deficiency(ies) on embryonic development is still largely unknown and warrants further investigation. The known specific defects of selected single and double knockouts will be discussed in subsequent sections. NO in reproduction/early preimplantation development NO participates in numerous biological processes associated with reproduction and embryogenesis (Gouge et al., 1998). Several studies have demonstrated a role for NO in ovulation, pregnancy, and preimplantation embryogenesis. Furthermore, multiple reproductive tissues, including the ovary, fallopian tubes, and uterus, express NOS isoforms. In the female reproductive tract, NO has been shown to play a role in ovulation and pregnancy through NOS inhibitor studies that resulted in inhibition of ovulation and inhibition/termination of pregnancy (Ekerhovd et al., 1997; Shukovski and Tsafriri, 1995; Telfer et al., 1995). Additionally, the oocyte and preimplantation embryo express NOS isoforms, suggesting a role for NO in oocyte meiotic maturation and early embryo development (Gouge et al., 1998; Purcell et al., 1999). Concomitantly, eNOS knockout mice exhibit defects in ovulation and oocyte meiotic maturation (Jablonka-Shariff and Olson, 1998). However, iNOS-derived NO has been suggested to play an inhibitory role during fertilization, suggesting isoform-specific reproductive affects (Yang et al., 2005). Furthermore, Gouge et al. demonstrated that NO was produced by preimplantation murine embryos and that embryos (2cell to blastocyst stage) cultured with a NOS inhibitor were developmentally delayed or nonviable. The authors speculated that NO participates in implantation and demonstrated for the first time that NO is required for early embryonic development in mice (Gouge et al., 1998). Subsequent studies, using both low and high concentrations of a NO donor (10−5 and 10−7 M), have also elicited inhibition of preimplantation embryonic development demonstrating the importance of maintaining the appropriate NO concentration, with too little or too much having significant effects on embryonic development (Sengoku

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et al., 2001). This tight regulation of NO during embryonic development is reminiscent of observations made in VEGF expression in hypo- and hyper-morphic transgenic mice, which also exhibit abnormalities in embryonic development (Miquerol et al., 1999, 2000). During the implantation process, NO has been demonstrated to be required for implantation by intrauterine administration of a NOS inhibitor. The trophoblast giant cells (TGCs) express eNOS and iNOS, and the trophoblast of the implanting blastocyst produces NO (Gagioti et al., 2000). NO mediates events during the invasion process at the fetal–maternal interface (Nathan and Xie, 1994; Novaro et al., 1997) through matrix remodeling (Chwalisz et al., 1996), vascular permeability, and inflammatory responses (Salvemini et al., 1993). Furthermore, in postimplantation embryos (E7.5), only the TGCs at the implantation site generate NO (Purcell et al., 1999), thus supporting the role of NO as a required mediator of blastocyst implantation at the uterine endometrium. NO in nervous system development NO has been demonstrated to play a role in the differentiation and development of several organ systems. eNOS/nNOS double knockout mice display delayed ipsilateral retinocollicular projection refinement (removal of inappropriately located axons), suggesting a role for nitric oxide as a repellent molecule during normal neuronal development (Wu et al., 2000). NOdependent synaptic plasticity has also been demonstrated in the chick ipsilateral retinocollicular projection, chick growth cones cultures, and frog retinal explant cultures. In addition to a role in regressive events in the CNS, NO affects neurotransmitter release and synaptic efficiency (Mize and Lo, 2000; Wu et al., 2000) as well as being required for postsynaptic differentiation of the embryonic neuromuscular junction (Bicker, 2005; Godfrey and Schwarte, 2003; Schwarte and Godfrey, 2004). NO has also been demonstrated to control the balance between cell proliferation and differentiation, thus controlling body size (Kuzin et al., 1996). In a later study, NO was found to affect the Rb signaling pathway, regulating entry into S phase of the cell cycle in the developing eye (Kuzin et al., 2000). NO in lung system development Given its importance in early developmental processes and the observation that NOS deficient mice exhibited increased neonatal mortality, potential later developmental NO-mediated processes including branching morphogenesis were investigated. Using a rodent day 13 fetal lung explant model, Young et al. demonstrated an effect of NO on pulmonary branching morphogenesis using both selected NO donors and NOS inhibitors, eliciting increased branching with exogenous NO donors and decreased branching with NOS inhibitors (Young et al., 2002). In a later study, Han et al. demonstrated that eNOS deficient mice exhibited major defects in lung morphogenesis including thickening of saccular septae, abnormalities in epithelial differentiation and airway development, reduced surfactant production, a paucity of distal arteriolar branches, capillary

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hypoperfusion, and misalignment of pulmonary veins in addition to congenital cardiac malformations (Han et al., 2004). The abnormal development of the distal arteriolar network was found to be similar to what was observed in a transgenic mouse expressing only VEGF A120 (Galambos et al., 2002), suggesting that NO may regulate angiogenic gene expression in the lung, which is known to have important effects on epithelial as well as endothelial development. The authors found these abnormalities to be very similar to the characteristics of alveolar capillary dysplasia in the human newborn population, illustrating the importance of NO regulation in human development and its effects in the neonatal period (Han et al., 2004). NO in the cardiovascular system In 1980's, NO was isolated as a factor that induces vasodilation and lowers blood pressure. Since then, it has been discovered to have numerous roles in endothelial cell and vascular biology, affecting migration, proliferation, apoptosis and tube formation, platelet activation and aggregation, platelet–luekocyte interactions, cell adhesion molecule expression, and tissue factor expression (Naseem, 2005). The generation of eNOS deficient mice significantly aided our understanding of the roles this NOS isoform and its product, NO, play in the development of the cardiovascular system. Though the eNOS KO mice are viable, perinatal, newborn, and adult demise is not infrequent and these mice display fetal growth retardation (Feng et al., 2002; Lee et al., 2000). eNOS deficient neonates display bicuspid aortic valves (Lee et al., 2000) and atrial and ventricular septal defects (Feng et al., 2002). Further investigations of eNOS wildtype, heterozygous, and null mice revealed direct relationships between the levels of eNOS and survival, cardiac fractional shortening, pulmonary congestion and septal defects, and inverse relationships between levels of eNOS and caspase-3 activity and myocardial and endocardial cushion apoptosis (Feng et al., 2002). In addition to these and many other murine-based studies, the complex roles of NOS and NO in cardiovascular development have been studied using the chick embryo. In a recent study, chronic NOS inhibition elicited impairment of endothelium-dependent relaxation and structural remodeling of the pulmonary vascular bed as well as biventricular increases in cardiac wall areas (Villamor et al., 2005). In addition, Feng et al. also observed an inverse relationship between eNOS levels and myocardial apoptosis/cell death in eNOS wildtype, heterozygous, and null mice (Feng et al., 2002). These data are consistent with the concept that eNOS expression levels (and thus NO production) are an important modulator of cardiac development, likely affecting the epithelial to mesenchymal transformation in the endocardial cushion areas, myocyte survival and myocardial angiogenesis and remodeling, and dysregulation of this NOS isoform in the heart resulting in structural congenital abnormalities and myocardial functional abnormalities (manifested by decreased fractional shortening), ultimately resulting in heart failure and increased mortality. These findings, coupled with data illustrating abnormal limb development in eNOS deficient mice (focal acute hemorrhages in the distal limbs and shortened peripheral digit bones), are

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consistent with the notion that altered NO generation may be at play in humans suffering with Holt–Oram (heart–hand) syndrome (Basson et al., 1994; Feng et al., 2002; Hefler et al., 2001). The role of eNOS and NO in cardiac myocyte development is complex, with NO playing roles in cardiomyogenesis and in myocardial angiogenesis (Bloch et al., 1999; Zhao et al., 2002). Specifically, murine embryonic hearts were found to express iNOS and eNOS starting at E8.5 (C-looped tubular heart stage). At later time points, cardiac iNOS expression was diminished, while only minor reductions in eNOS were noted, suggesting the participation of specific NOS isoforms in the process of cardiomyogenesis (Bloch et al., 1999). Interestingly, ectopic iNOS expression and activity accompany various diseases in the cardiovascular system. Induction of the high flux iNOS enzyme is believed to play a role in the pathophysiology of these diseases, while the low flux eNOS enzyme serves a protective function (Gewaltig and Kojda, 2002; Shah, 2000). The importance of NOS activity and NO production during cardiomyogenesis was confirmed using murine ES-cell-derived cardiomyocytes. Treatment of these ES-cell-derived cardiomyocytes with NOS inhibitors resulted in decreased development of distinct cross-striation patterns in the cells, consistent with a block in maturation, and addition of NO donor reversed this effect. These studies raised the possibility that a potentially direct role for NO in cardiomyocyte maturation could partially explain the development of heart failure observed in eNOS deficient mice. Regarding the diminished myocardial angiogenesis observed in eNOS deficient mice, Zhao et al. (2002) suggested that the diminished production of NO in eNOS deficient mice would alter VEGF signaling as well as the persistence of VEGF expression (as the angiogenic effect of VEGF is thought to be largely mediated via eNOS and NO itself is an upstream promoter of VEGF expression; Papapetropoulos et al., 1997a,b). The decreased myocardial angiogenesis in eNOS deficient mice could very well contribute to myocardial apoptosis, progressive heart failure, and increased mortality by resulting in the formation of an insufficiently vascularized vascular bed, leading to chronic ischemia. Furthermore, abnormal angiogenesis and capillary development in various vascular beds could very well be the root cause of the moderate systemic hypertension, mild elevation in pulmonary vascular resistance, and the exaggerated pulmonary vasoconstrictor response to hypoxia noted in the adult animals (Zhao et al., 2002). In addition, in vivo studies have suggested a role for eNOSderived NO in the closure and remodeling of the ductus arteriosus, the fetal vascular shunt between the aorta and pulmonary artery (Richard et al., 2004). Preterm administration of LNAME to pregnant female rats on E19 resulted in increased ductal constriction in the fetuses (Takizawa et al., 2000). NO in vitelline vasculogenesis The importance and complexity of NO production and signaling are manifested in the development of the vitelline vasculature. Establishment of the extra-embryonic vasculature is crucial for survival, growth, and homeostasis in the vertebrate embryo. The use of whole conceptus culture has been important

in facilitating our understanding of the roles of selected growth factors (Pinter et al., 2001), cell adhesion molecules (Enciso et al., 2003; Pinter et al., 1997, 1999, 2001), and enzymes (Nath et al., 2004) during normal and hyperglycemia-induced dysregulated vitelline circulation and endocardial cushion development. Using this model (Figs. 1A–D), we have determined

Fig. 1. The dynamic localization and expression of NOS isoforms during the development of the vitelline circulation. (A–D) In vitro culture of murine conceptuses from 7.5 dpc (A, B) to 9.5 dpc (C, D). Illustrated is the development of the vitelline vasculature and organogenesis. The yolk sac is observed to consist of an endodermal layer (En) overlying a layer of mesoderm (Mes), from which angioblasts will differentiate, which will further differentiate into the endothelia of the yolk sac vasculature (V). (E–J) Expression pattern of NOS isoforms during three stages of vascular development. Conceptuses were excised from timed pregnant matings at 8.0 dpc (A, D), 8.5 dpc (B, E), and 9.5 dpc (C, F) which correspond to the blood island formation, primary capillary plexus, and vessel maturation stages, respectively. Immunofluorescence for eNOS (E– G) and iNOS (H–J) was performed (red = NOS, blue = DAPI). Images of single blood islands or vessels stained with NOS were captured at 40× magnification and merged with DAPI images. En = endoderm and Me = mesoderm/endothelium (n ≥ 4) (data derived from Nath et al., 2004). (K and L) Temporal distribution of endogenous NOS isoforms during vascular development. (K) Graph of Erk-2 normalized data for NOS protein expression in pooled in vivo grown 7.5 conceptuses and 8.5 and 9.5 yolk sacs. The dashed line represents eNOS, while the solid black line represents iNOS (n = 4, P b 0.05, P b 0.01). The quantified signals represent mean ± SEM. The relative expression levels were compared to 7.5 dpc levels. (L) Representative Western blot of the graphed data (data taken from Nath et al., 2004).

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that there is a robust modulation of NOS isoform expression in both the endodermal and mesodermal compartments of the yolk sac during the stages of vasculogenesis through remodeling and arborization of the vasculature (Figs. 1E–J) (Nath et al., 2004). Namely, iNOS expression appears localized in the endodermal compartment at E8.0 and is non-detectable at later time points, while eNOS is initially present in the mesodermal compartment in low amounts at E8.0, but its expression increases in the differentiating endothelial cells at E8.5 and E9.5. In addition to the changing localization patterns of iNOS and eNOS during yolk sac vascular development, their protein levels were also noted to change, with iNOS progressively decreasing from E7.5 to E9.5, while eNOS was noted to progressively increase during this time period (Figs. 1K and L). This inverse relationship in iNOS and eNOS expression has been noted during cardiac development albeit at a different time period of development (E14.5) (Bloch et al., 1999), but it suggests a similar dynamic NOS isoform relationship, perhaps being indicative of a general switch in isoform expression during distinct differentiation processes. Interestingly, the early robust expression of iNOS in the endodermal compartment is associated with robust endodermal NO production in the conceptus (Nath et al., 2004). This is followed temporally by an increased expression of VEGF at E8.5 (Madri et al., 2003), consistent with the notion that NO is an upstream promoter of VEGF expression (Jozkowicz et al., 2001, 2004). This concept is supported by data accrued by Han et al. in which they observed that mRNA levels of VEGF-A and angiopoietin-1 were reduced in eNOS null mice and these mice exhibited impaired pulmonary angiogenesis. Interestingly, this pulmonary vascular pathology mimics a disease entity found in the human newborn population, human alveolar capillary dysplasia (Han et al., 2004), again suggesting that dysregulation of NO could be a cause of a human vascular disease. Additional targets of NO during the development and differentiation of the vitelline and other vasculatures are currently unknown and warrant investigation. Dysregulation of NO in the yolk sac by hyperglycemia: a model explaining increased congenital abnormalities in the offspring of diabetic mothers? Offspring of diabetic mothers (both humans and experimental animals) experience a two- to four-fold increase in congenital anomalies (Eriksson et al., 1991; Kitzmiller et al., 1996). Although no particular organ system or tissue appears to be specifically targeted, a variety of cardiovascular anomalies are frequently observed and comprise one of the more common birth defects (1% of live births) that carry a significant mortality (21%) (Ferencz et al., 1990). In addition to the increased incidence of congenital anomalies noted in the live births and stillborns of diabetic mothers, these mothers experience difficulties in becoming pregnant and exhibit an increased incidence of fetal resorption (Baker and Piddlington, 1993; Ellington, 1997; Eriksson et al., 1991; Pampfer et al., 1997; Schwarz and Teramo, 2000). Following implantation and before placentation, embryonic growth is dependent upon proper develop-

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ment of the yolk sac vasculature and the vitelline circulation. Arrested or mal-development of this vasculature would lead to fetal demise at early gestation contributing to difficulty in maintaining pregnancy, while arrest and/or mal-development of vasculature(s) at later times, associated with specific organ/ tissue development, would contribute to congenital abnormalities in a wide variety of organs/tissues. It has previously been shown that in vitro cultures of rat and mouse conceptuses at the primitive streak stage maintain viability and the conceptuses develop near normally for 48 h, during the initial stages of organogenesis. During this period, they are nourished via the vitelline circulation (Pinter et al., 1986, 1999) which initially develops prior to the onset of organogenesis. The addition of 20 mM D-glucose (a plasma concentration of glucose often observed in diabetic mothers (both in humans and experimental animals) to these cultures results in significant yolk sac and embryonic vasculopathy and abnormal embryonic development (Pinter et al., 1999). Recently, we have demonstrated that VEGF expression is dysregulated in these D-glucose-treated embryos and in the offspring of streptozotocin-induced diabetic pregnant mice (Madri et al., 2003; Pinter et al., 1999, 2001). In light of these findings and data illustrating crucial roles of several growth factors and their receptors during early vasculo- and angiogenesis (Tammela et al., 2005), we investigated whether NOS isoform expression, activities, and NO production was modulated in response to hyperglycemic insult and whether hyperglycemia-induced yolk sac vasculopathy could be rescued by addition of exogenous NO donors. Interestingly, we have noted that this glucose-induced vasculopathy is correlated with the persistence of iNOS expression and a reduction in eNOS expression and could be reversed by the addition of an exogenous NO donor, which correlated with a correction of both iNOS and eNOS relative expression levels, implicating NO as a crucial modulator of early vasculogenesis (Madri et al., 2003; Nath et al., 2004; Pinter et al., 1999, 2001). Additionally, exogenous addition of L-NMMA arrested yolk sac vascular development at the primary capillary plexus stage (not shown) as does hyperglycemic insult. The rescue of the vitelline vasculo- and angiogenesis process by exogenous NO donor suggests the possible future development and potential therapeutic use of NO donors in the treatment of maternal diabetes-induced embryonic vasculopathy. While dysregulation of NOS and NO has been implicated as being intimately involved in the development of diabetic vasculopathy (Naseem, 2005), our understanding of the specific mechanisms involved is still incompletely appreciated. One area that has received considerable attention is the increased production of ROS (reactive oxygen species) in hyperglycemia (resulting in cellular toxicity), leading to a depletion of NO (resulting in dysregulation of NO signaling) during embryonic development (Brodsky et al., 2001; Mügge et al., 1991; Urbich et al., 2002; Zhang et al., 2001). Indeed, conceptuses cultured under hyperglycemic conditions exhibit increased ROS, while similar cultures treated with an exogenous NO donor exhibit normal vascular development and exhibit a partial decrease in ROS expression, consistent with NO's antioxidant/scavenging

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capabilities (Nath et al., 2004). Similar to our findings, Wells et al. demonstrated that iNOS knockout mice are less susceptible to embryopathy by ROS-inducing agents during organogenesis, a teratogen-sensitive stage (Kasapinovic et al., 2004). An emerging area that may shed considerable light on the underlying mechanism(s) at play in hyperglycemia-induced embryonic vasculopathy is the potential dysregulation of protease activities by alteration of NO levels (Akool et al., 2005; Chen and Wang, 2004; Phillips et al., 2001; Phillips and Birnby, 2004). These investigators have demonstrated several distinct NO-dependent pathways which, when NO levels are altered, result in modulation of MMP-2, MMP-9, and TIMP-1, with increased NO driving either: (1) decreased caveolin-1 levels which in turn are associated with decreased MMP-9 expression; (2) increased expression of the transcription factor ATF-3, which in turn elicits a reduction in p53, leading to a reduction in MMP-2 expression; and/or (3) increased TGF-βmediated Smad pathway induction of TIMP-1, which in turn would inhibit MMP-9. Thus, dysregulation of NO levels resulting in alteration of either ROS-mediated cell motility/migration (Gratzinger et al., 2003; Ushio-Fukai and Alexander, 2004) and/or MMP expression (Akool et al., 2005; Chen and Wang, 2004; Phillips et al., 2001; Phillips and Birnby, 2004) would have profound effects

on development of the vitelline circulation (Nath et al., 2004) and endocardial cushion formation (Enciso et al., 2003) (Fig. 2). Given the advances in gene targeting and delivery, small molecule design and delivery and in optimizing strategies that appropriately scavenge increased ROS and/or restore NO levels and signaling during early embryonic development (organogenesis), NOS/NO-based therapies are becoming approachable and with more investigation have the potential of developing into beneficial therapeutic approaches for cardiovascular and other diseases in the future (Channon et al., 2000; Chen et al., 2002). Conclusions As mentioned throughout this text, NOS isoforms and their product, NO, are important (critical) modulators of developmental processes, ranging from the preimplantation through the perinatal period. In spite of intense investigation, many of the process- and stage-specific targets of NO and their up- and down-stream signaling cascades are incompletely elucidated. With the elucidation of the mechanisms at play during specific stages of development and during the development of particular organs/tissues will come the possibility to intervene favorably in several NO-mediated processes that are known or suspected to be dysregulated in several disease states.

Fig. 2. Potential mechanisms of hyperglycemia-induced NO-mediated arrest of vitelline circulation and endocardial cushion development. (A) NO modulation of ROS levels may affect lamellipodia formation and motility/migration of endothelial cells, arresting vascular remodeling of the yolk sac primary capillary plexus and epithelial to mesenchymal transformation in endocardial cushions. (B) NO modulation of MMP cellular localization (by altering Cav-1 expression), expression (by altering transcription via ATF-3 and p53), and/or activity (by altering TIMP-1 levels) may affect vascular remodeling of the yolk sac primary capillary plexus and epithelial to mesenchymal transformation in endocardial cushions. (a, b, c and d) Representative micrographs of murine 9.5 dpc conceptuses cultured under normoglycemic conditions (a and b) or hyperglycemic conditions (c and d), illustrating a well-developed arborizing, mature vitelline vasculature in conceptuses grown under normoglycemic conditions (5 mM D-glucose) (a and b) and an abnormal vitelline vasculature comprised of ecstatic vessels displaying no arborization, consistent with a vasculature arrested at the primary capillary plexus stage in conceptuses grown under hyperglycemic conditions (20 mM D-glucose) (c and d). Figure based on data from Akool et al. (2005), Chen and Wang (2004), Phillips et al. (2001), Phillips and Birnby (2004), Gratzinger et al. (2003), Ushio-Fukai and Alexander (2004), Pinter et al. (1997), Pinter et al. (1999), Pinter et al. (2001), Nath et al. (2004), and Enciso et al. (2003).

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