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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Free Radic Biol Med. Author manuscript; available in PMC 2012 December 15.
Published in final edited form as:
PMCID: PMC3232180
NIHMSID: NIHMS330450

Nitric oxide-cyclic GMP signaling in stem cell differentiation

Abstract

The nitric oxide-cyclic GMP (NO-cGMP) pathway mediates important physiological functions associated with various integrative body systems including the cardiovascular and nervous systems. Furthermore, NO regulates cell growth, survival, apoptosis, proliferation and differentiation at the cellular level. To understand the significance of the NO-cGMP pathway in development and differentiation, studies have been conducted both in developing embryos and stem cells. Manipulation of the NO-cGMP pathway by employing activators and inhibitors as pharmacological probes and/or genetic manipulation of NO signaling components has implicated the involvement of this pathway in regulation of stem cell differentiation. This review will focus on some of the work pertaining to the role of NO-cGMP in differentiation of stem cells into cells of various lineages particularly into myocardial cells and stem cell based therapy.

Keywords: Nitric oxide, cyclic GMP, stem cells, differentiation

Introduction

Nitric oxide (NO) is a diatomic free radical and one of the most important signaling molecules in mammalian physiology. NO mediates a host of physiological events such as smooth muscle relaxation, vasodilatation, neurotransmission, inhibition of platelet aggregation, and immunmodulation [1-3]. Furthermore, at the cellular level, NO regulates cell growth, survival, apoptosis, proliferation and differentiation [3]. NO is also involved in the pathophysiology of several diseases such as diabetes, cancer, cardiovascular disease and various inflammatory conditions due to its reduced bioavailability, production or due to overproduction of NO [2]. NO is synthesized by a group of enzymes known as nitric oxide syntheses (NOS) by oxidation of a guanidino nitrogen of L-arginine into L-citrulline and NO [3]. There are 3 isoforms of NOS (NOS-1, NOS-2 and NOS-3) which utilize a complicated array of co-factors and co-substrates such as molecular oxygen, NADPH, tetrahydrobiopterin (BH4), flavin mononucleotide, flavin adenine dinucleotide, heme and calcium/calmodulin for the synthesis of NO [2-3]. NOS-1 (neuronal NOS) and NOS-3 (endothelial NOS) are constitutive NOS enzymes regulated by calcium/calmodulin. NOS-2 enzyme (inducible NOS) is induced in response to inflammatory stimuli and is activated independent of calcium as the enzyme is always bound to calcium/calmodulin once it is synthesized. The three isoforms of NOS are encoded by three different genes on different chromosomes and they exhibit 50-60% identity with each other and cytochrome p450 enzymes [4-7] while the identity of a particular isoform between species can be between 85-90%. The NOS isoforms have a heme prosthetic group and are active as homo-dimers consisting of two identical monomers which can be divided into a C-terminal reductase carboxy domain and an N-terminal oxygenase amino domain [5, 8].

NO mediates its downstream effects either through cyclic GMP (cGMP) dependent or independent mechanisms. The cGMP dependent mechanism is mediated through its receptor soluble guanylyl cyclase (sGC). cGMP independent effects of NO are mediated through its interaction with metal complexes, oxygen (O2) and superoxide (O2 ·) that mediate various downstream events. Interaction of NO with oxygen species leads to nitrosation and nitration of proteins. These posttranslational modifications particularly occur during an inflammatory response and in cases where superoxide dismutase is not able to scavenge excess superoxide (O2 ·) [1].

This review is particularly focused on demonstrating how manipulation of the NO/cGMP pathway is involved in regulation of stem cell differentiation. In addition to discussing our contributions to the stem cell field pertaining to NO/cGMP, we have included major findings wherever possible. We do apologize to some investigators for the omissions made, as it is not possible to include more than 120, 000 publications on the NO-cGMP pathway.

Guanylyl cyclase and cGMP

The second messenger cGMP is synthesized by a family of enzymes known as guanylyl cylases that share amino acid sequence with the catalytic domain of the adenylyl cyclases which synthesize another second messenger, cyclic AMP [9]. Two classes of guanylyl cyclases (soluble and particulate) synthesize cGMP from intracellular guanosine triphosphate (GTP). Particulate cyclases (pGC) are membrane bound receptors that bind to ligands such as natriuretic peptides, guanylin peptides and E. coli heat stable enterotoxin (STa).

Soluble guanylyl (sGC) is the only known physiological receptor of NO. sGC is a heme containing obligatory hetero-dimer composed of α (~ 80 kDa) and β (~ 70 kDa) subunits which make up the active enzyme. To date, four sGC isoforms (α1, α2 and β1, β2) encoded by four different genes have been identified. However, only sGC α11 and sGC α21 heterodimers are activated by NO [2-3, 10]. sGC α11 are ubiquitously expressed in adult mammalian tissue with the highest levels in the brain, lung, heart, kidneys, muscle and spleen [11]. Furthermore, vascular smooth muscle and endothelial cells also predominantly express α1 and β1 subunits [12]. However, in addition to sGC α11 the brain exhibits the highest expression of sGC α21 form as well. The formation of α21 heterodimer was also identified in the human placenta [10].

Each sGC subunit consists of three domains which make up its structure and function: a) an N-terminal heme-binding domain; b) a dimerization domain and c) a C-terminal catalytic domain. At the functional level, NO binds to the heme prosthetic group of its physiological receptor sGC, which catalyzes the conversion of GTP to the second messenger cGMP. NO mediates most of its physiological effects as a cell signaling molecule through the production of cGMP by sGC. cGMP directly interacts with its downstream effectors such as cGMP dependent kinase (cGKI or PKG), cyclic nucleotide gated channels (CNG) and cGMP dependent phosphodiesterase (PDEs) to mediate physiological effects such as vascular smooth muscle tone and motility, phototransduction, and maintaining fluid and electrolyte homeostasis [13-15]. The genomic structures and promoters of sGC α1 and sGC β1 subunits and transcriptional regulation of sGC expression have been previously described [16-17].

In response to NO, sGC activity increases more than 200 fold; however, this stimulated activity is quickly removed by phosphodiesterase (PDE) 5A enzyme [18-19]. Previous studies suggest that gene expression of sGC α1 and β1 subunits decrease during aging and hypertension and the mRNA levels of all subunits vary during the embryonic development [20]. In addition to NO donors [21], estrogen [17], cAMP elevating compounds, [22-23] and cytokines [24] also modulate the expression of sGC. Alternative splicing of sGC α and β subunits may reveal another regulatory mechanism of sGC activity. For instance, the sGC α2i splice variant carries an insertion in the catalytic domain. This variant was detected in some human tissues and it was shown to exhibit a dominant negative function [25]. Alternative splice forms of the β1 and β2 subunit have also been reported [26-28]. Furthermore, three new sGC α1 splice variants (N1-sGCα1, N2- sGCα1 and C-sGCα1) in different human tissues were identified. Functional analysis revealed that N1-sGCα1 protein demonstrated a dominant negative effect on the activity of sGC where as C-sGCα1 produced a fully functional NO sensitive enzyme [29].

Allosteric activators of sGC

Previous studies suggest that the loss of NO bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Although NO donors and organic nitrates provide symptomatic improvement in patients with cardiovascular disease, there is no evidence that such treatment can reduce mortality. Therefore, it is proposed that NO independent heme dependent sGC stimulators and NO independent and heme independent sGC activators might provide an advantage over the currently employed therapies [30]. Previous studies suggest that sGC stimulators such as YC-1, BAY41-2272, and BAY41-8543 can be used as pharmacological tools to stimulate the activity of sGC under reduced heme moiety in many cell systems. Furthermore, combination of NO donors and sGC stimulators induce cGMP accumulation in a synergistic manner [30]. Allosteric sGC stimulators, BAY41-2272 and YC-1 are reported to also inhibit PDE 5 activity, an enzyme which degrades cGMP. The second group comprise of NO independent and heme independent sGC activators (BAY 58-2667 and HMR-1766) which demonstrate potent action on the oxidized form of sGC enzyme [30, 31].

Effectors of sGC

sGC mediates many of its physiological effects through production of cGMP. One of the major targets of cGMP is the cGMP dependent protein kinase, PKG. The binding of cGMP to PKG leads to its activation as a serine/threonine kinase [13]. Activated PKG can act on many substrates including the IP3 receptor, phosholamban, vimentin, the phosphatase inhibitor G substrate and subunits of myosin light chain phosphatase [32-37]. There are two distinct genes that encode PKG (I&II) with PKGI resulting into two isoforms (PKGIα and PKGIβ) through splicing. These isoforms are differentially expressed in various tissues and are activated by various stimuli [38-42]. Studies have shown that PKGIα directly phosphorylates myosin phosphatase, an enzyme that regulates vascular smooth muscle tone [37].

Cyclic nucleotide gated channels (CNG) are another target of cGMP. CNGs regulate Na+ and Ca+ influx into cells. Activation of CNG by cGMP is produced by retinal guanylyl cyclase (particulate enzymes) which regulates phototransduction and neurotransmission in the retina [13-14].

The cyclic nucleotide phosphodiesterases (PDE) are a family (PDE1-11) of enzymes that either hydrolyze second messengers, cAMP and cGMP and/or are regulated by cGMP [43]. cGMP regulates the activity of PDE-2,-3,-5 and -9. Out of these, PDE-2, -3 and -5 are expressed in cardiac myocytes, PDE-3 and -5 in vascular smooth muscle cells, and PDE-2,-3 and -5 in vascular endothelial cells [43-50]. PDE-2 can hydrolyze both cAMP and cGMP, whereas PDE-5 selectively hydrolyzes cGMP. PDE-5 is a cytosolic protein with a particular localization in Z-bands of cardiac myocytes suggesting its association with other Z-band proteins such as PKG [51-52].

Role of Nitric Oxide-cyclic GMP in Development

Previous studies clearly demonstrate the crucial role of NO during development. This role has been mainly identified by the creation of NOS knockout animals in all combinations of the three isoforms [53]. Previously, the ability of all single NOS knockout models to develop rather normally, although with heart malformations, was attributed to compensation by the other NOS isoforms [54-55]. Double knockout animals in all cases exhibited decreased viability and the inability to produce offspring with other double knockouts. However, since those double knockouts were able to reproduce with wildtype animals, a defect in development was evident. Triple NOS knockout animals which completely lack endogenous production of NO also demonstrate low survival rate and significant reduction in number of offspring [56]. The animals present with nephrogenic diabetes insipidus and arteriosclerosis. These results provide the first evidence that the systemic deletion of all three NOSs can cause a variety of cardiovascular diseases in mice, thereby demonstrating the critical role of the endogenous NOS system in maintaining cardiovascular and volume homeostasis.

Further significance of NO-cyclic GMP pathway in development has emerged from generation of knockout animal models of other components of the NO signaling pathway. Previous work using chemical inhibitors also demonstrated a role of NO/cGMP in preimplantation embryo and apoptosis in the mouse [57]. Similarly, the functional significance of sGC α11 heterodimer was demonstrated by conducting studies with sGC α1 knock out animals. sGC α1 knockout mice of both genders exhibit significantly decreased relaxing effects of major vasodilators such as acetylcholine, NO, YC-1 and BAY 41-2272 [58]. Similar results were demonstrated with cGMP dependent protein kinase I (cGKs) and sGC β1 knockout animals [59-60]. The significance of PKG in development can be evaluated by studying PKG knock out (cGKI) animals, which show impaired NO/cGMP-dependent vasorelaxation and blunted cardiac negative inotropic response to cGMP along with a host of other problems in cardiovascular and neuronal systems [61]. sGC β1 knockout animals exhibit the most severely developmental phenotype. About 70-80% sGC β1 knockout animals died within two days after birth and 90% of survivors died within two weeks of severe gastrointestinal abnormalities [59]. These findings confirm the essential role of sGC-cGMP-cGKI in mediating various effects of NO signaling components in vivo.

Stem cells

Adult stem cell transplantation to treat cardiovascular, neurodegenerative and other diseases have been studied for many decades [62]. Although adult stem cells have been successfully used for allogenic transplantation for treating patients with hematological malignancies and a variety of inherited or acquired immune-hematological diseases [63], their limited proliferative capacity and plasticity limit their use in regenerative medicine. Furthermore, studies suggest that improved cardiac function due to bone marrow transplant has been mainly attributed to the paracrine action from transplanted cells [64]. Therefore, use of embryonic and induced pluripotent stem cells could potentially revolutionize the field of regenerative medicine albeit with some disadvantages. Embryonic stem (ES) cells derived from the inner cell mass of blastocysts exhibit two unique properties 1) self-renewal which allows cells to grow continuously. (2) Pluripotency is a unique signature property which allows ES cells to differentiate into the derivatives of all the three germ layers; ectoderm, mesoderm and endoderm [64-65]. ES cells have been successfully differentiated into cells of many lineages including cardiac lineage by directed differentiation, embryoid body (EB) directed differentiation, and stromal cell co-culture methods [64].

Induced pluripotent stem (iPS) cells were initially generated by Yamanaka and colleagues by identifying a set of key transcription factors (OCT4, SOX2, KLF-4 and c-myc) which can reprogram somatic cells such as fibroblasts to a pluripotent state when over-expressed [66]. Further studies indicated the possibility of re-programming mouse or human somatic cells using a different set of 4 factors (OCT4, SOX2, NANOG and LIN 28) [67-73]. The recent discovery of a new pluripotency factor, RONIN, may also facilitate the production of iPS cells from human extraembryonic tissues through its co-expression with KLF-4 and c-myc [74-75]. Somatic cells have also been induced to a pluripotent state by introduction of only two transcription factors (OCT4, SOX-2) and valproic acid (VPA), a small molecule histone deacetylase inhibitor (HDAC) which increases the efficiency of reprogramming primary human fibroblast to pluripotent state [76]. Earlier studies also indicate that iPS cells can be differentiated into cells of various lineages such as myocardial and neuronal cells with similar approaches as ES cells. [77-79]. Although, iPS cells appear to be similar to ES cells with respect to morphology, global gene and protein expression and differentiation potential; more recent studies suggest that iPS cells do differ from ES cells in gene expression, DNA methylation state and miRNA expression [80-82].

Role of NO in Stem Cell Differentiation

The significant role of the NO-cGMP in regulation of stem differentiation is beginning to emerge from our studies [83-89] and that of other investigators. The significance of the NO-cGMP pathway in the differentiation of adult stem cells is indicated by the work of other investigators which show that alteration of intracellular NO-cGMP levels can modulate the differentiation of bone marrow derived progenitor cells [90-94]. Previous studies indicate that the exposure of CD34+ bone marrow derived cells to NO donors shows inhibition of erythroid but induction of myeloid colony formation [90]. Furthermore, NO production by murine bone marrow cells was shown to inhibit the colony formation ability of isolated Sca-1 and Thy-1 positive cells [91]. The importance of NO in regulation of stem cell number and function in vivo was demonstrated by exposure of mice to NOS inhibitors which showed an increase in the number of stem cells compared to the untreated mice [92]. Further studies conducted in eNOS knockout mice demonstrate impaired mobilization in BM stem and progenitors cells [93] that may be related to impaired neo-vascularization. Moreover, eNOS knockout mice exhibit markedly reduced capacity to produce endothelial progenitor cells from hematopoietic stem cells. Therefore, these mice are unable to re-vascularize the tissue subjected to ischemic injury [94].

The role of NO in heart development has been implicated by studies in both developing embryos and ES cells. For instance, both NOS-2 and NOS-3 were detected during the early stages of cardiomyogenesis in mouse embryos. ES cell-derived cardiomyocytes, in the same study, were also shown to express NOS-2 and NOS-3, similar to that seen in vivo. Incubation of these cultures with the NOS inhibitor L-NAME resulted in a profound differentiation arrest of these cardiomyocytes [95]. It was further demonstrated that NO can facilitate ES cell-derived cardiomyogenesis through both exogenous stimulation with NO donors or by introduction of the NOS-2 gene. Interestingly, the role of endogenous NO in the differentiation of ES cells into cardiomyocytes from this study appeared to differ from the previous, since the differentiation was only slightly compromised in the presence of L-NAME [96].

Role of sGC in stem cell differentiation

Our studies with mouse and human embryonic stem (ES) cells demonstrate differential expression and function of NOS isoforms, sGC subunits and PKG during differentiation of stem cells into cells of myocardial lineage (Table 1). Our studies indicate that the NOS-1 isoform is expressed in both mouse and human ES cells and mRNA and protein levels fall to basal level as ES cells differentiate. On the contrary, we observed a temporal induction of both NOS-2 and NOS-3 isoforms during various stages of ES cell differentiation in murine and human ES cells. Furthermore, our studies indicate that ES cells do not express enzymatically active NO receptor sGC. However, sGCα1, α2 and β1 levels increase during cell differentiation that coincides with a robust increase in NO-inducible intracellular cGMP levels in ES-derived differentiated cells and cardiomyocytes [83-84]. In contrast, sGC β2 gene expression in human ES cells declines as cells progress through differentiation, indicating that this subunit may have some role in the pluripotency of stem cells.

Table 1
Regulation of nitric oxide signaling components during mouse and human embryonic stem cell differentiation

We have also demonstrated that stem cells express low levels of protein kinase G. However, differentiated cells show robust temporal induction of PKG protein levels [83]. These results collectively point to the involvement of NO signaling components in the differentiation of stem cells. Furthermore, we demonstrated for the first time that in addition to NO donors, allosteric sGC stimulators BAY 41 2272 and YC-1 markedly increased the expression of cardiac specific transcription factors and cardiac specific proteins [85]. A combination of various NO donors and sGC stimulators further enhanced the expression of cardiac specific markers with a concomitant increase in the expression of sGC subunits at mRNA and protein level. cGMP analysis in undifferentiated cells revealed a lack of stimulation with NO donors. However, ES-derived differentiated cells acquired the ability to be stimulated with NO donors and sGC activators. These studies therefore demonstrate that NO donors and various sGC activators alone or in combination may facilitate differentiation of stem cells into myocardial cells with the robust increase in second messenger cyclic GMP (cGMP) accumulation [85], thereby implicating an important role of NO/cGMP pathway in differentiation of stem cells. Our unpublished studies suggest that in contrast to ES cells, there may be an aberration in the NO/cGMP pathway in iPS cells. We are further characterizing this in current studies.

The NO receptor sGC is regulated at multiple levels. sGC has been shown to be regulated at transcriptional, post transcriptional, and post translational levels [29,97-100]. At the post-transcriptional levels alternative splicing produces a number of sGC splice variants [25-27, 29, 101]. Previous studies have established the presence of sGC α1 splice variants, which encode proteins with deletions in both the C- and N-termini [29]. One such characterized splice form (sGC- α1N1) exhibited dominant negative properties while another splice variant (sGC-α1C) formed a fully active heterodimer with the β1 subunit. Since sGC alternative splicing has been shown to regulate sGC function, we reasoned that it might also play a role in stem cell differentiation by modulating sGC activity. Accordingly, our recent studies demonstrate that sGCα1 subunit undergoes alternative splicing during ES cell differentiation [86]. The C-type sGC α1 splice variant is highly expressed in differentiating cells; however, its intracellular distribution varies from the canonical sGCα1 subunit [86].

NO-cGMP pathway has also been implicated in the differentiation of stem cells into cells of various lineages in response to various plant compounds. Zhu et al [102] demonstrated that icariin (a constituent of Epimedium, a traditional Chinese medicine) induced differentiation of mouse ES cells into cardiomyocytes by elevation of cAMP/cGMP ratio in ES cells as well as upregulation of the endogenous generation of NO during the early stages of cardiac development. Similarly, the plant compound genistein has been shown to stimulate osteoblastic differentiation in bone marrow culture via the NO-cGMP pathway [103]. Our most recent studies indicate that polyphenol curcumin induces differentiation of stem cells via modulation of the NO pathway [104].

In order to study the mechanism of NO induced differentiation of stem cells, a recent study [105] demonstrated that the NO donor DETA-NO induced differentiation of mouse and human ES cells by downregulation of two master genes Nanog and Oct-4. Exposure of cells to DETA-NO along with valproic acid further enhances the differentiation of stem cells into cells of endodermal lineage [105]. Another study suggests that exposure of mES cells to low concentrations of NO promotes their survival by the inhibition of pro-apoptotic and up regulation of anti-apoptotic genes [106].

Role of PKG in stem cell differentiation

The significance of PKG in development can be evaluated by studying PKG knock out (cGKI) animals, which show impaired NO/cGMP-dependent vasorelaxation, blunted cardiac negative inotropic response to cGMP, along with a host of other problems in cardiovascular and neuronal systems [61]. Studies from our laboratory indicate absence of PKG expression in mouse stem cells. However, PKG levels increased as cells went through differentiation process [83] suggesting a possible role of PKG in differentiation. Our previous studies [85] have also shown that cGMP analog 8-bromo-cGMP, which activates PKG, can induce differentiation of stem cells. Studies from other investigators have also indicated that PKG can positively regulate the migration of neural precursor cells [107] and survival of OP9 progenitor cells [108]. A recent study demonstrated that cGMP modulates the fate of neural stem cells in vivo as higher GMP levels differentiate the neural stem cells to neuronal cells [107]. In contrast, downregulation of PKG and PKC has been shown to enhance cardiomyocyte production during ES stem cell differentiation [109]. Therefore, it seems that PKG might play an important role in regulation of stem cell differentiation and possibly in proliferation and survival of stem cell-derived cardiomyocytes.

Role of PDEs in stem cell differentiation

The number of circulating blood progenitor cells is reduced in patients with cardiovascular risk. Previous studies have shown that PDE5 inhibitor Vardenafil increases circulating progenitor cells in humans possibly due to increased cGMP levels [110]. Another study has shown that PDE7 may play an important role in osteoblastic differentiation [111]. Administration of PDE inhibitor Sildenafil has been shown to differentiate neural stem cells into neurons in brain in vivo [107]. Cyclic GMP has been shown to prevent heart failure induced by hypertrophy and pathological remodeling. Earlier studies have shown that (by blocking the catabolism of cGMP) PDE5A inhibitor Sildenafil Citrate suppresses chamber and myocyte hypertrophy in mice suggesting that PDE5A inhibition may provide a novel treatment strategy for cardiac hypertrophy and remodeling [112].

Role of Nitric oxide-cGMP in heart and in stem cell based therapy

The role of NO in regenerative potential of ESC cells was recently shown in a mouse model of hindlimb ischemia where NO treated ESC injected in the cardiac left ventricle selectively localized in the ischemic hindlimb and contributed to the regeneration of muscular and vascular structures [113]. Transplantation therapy using stem cells has a promise to revolutionize regenerative medicine. ES cell-derived cardiomyocytes show great promise because a) Unlimited self-renewal properties of ES cells could theoretically provide an unlimited supply of cardiomyocytes [65], b) ES cells can reliably be differentiated into cardiomyocytes in vitro, avoiding the plasticity controversy of adult stem cells [114-116], and c) ES cell-derived cardiomyocytes may escape xenograft rejection, even in the absence of immunosuppressive therapy [117-120]. It has been previously demonstrated that ES cells delivered to the heart after damage do engraft and improve heart function [117,120]. Furthermore, when engrafted ES cells were engineered to express vascular endothelial growth factor (VEGF), heart improvement was enhanced which may be due to increased formation of capillaries and blood delivery [121].

NO signaling plays an integral role in heart function, including ventricular compliance, myocardial preload, afterload, reperfusion injury and angiogenesis [122-125]. Following myocardial infarction, NOS-2 expression is increased, which may contribute to myocardial dysfunction and injury [126-127]. In contrast, production of NO by NOS-1 and NOS-3 could be beneficial for the heart following myocardial infarction [128-129]. It seems that these beneficial effects of NO occur through a cGMP-mediated pathway, since NO and cGMP can protect cardiomyocytes in hypertrophy models [130-131]. This protection was later suggested to occur through activation of PKG [132]. Therefore, understanding NO signaling in the context of the derivation and delivery of ES cell-derived cardiomyocytes and transplantation may further optimize the conditions for repair following myocardial infarction.

Notably, eNOS-derived NO has been shown to increase the beneficial production of VEGF, implicating a novel therapy for cardiovascular diseases [133]. Previous studies have shown that hyperstimulation of cGMP synthesis via activation of PKG or genetic upregulation of natriuretic peptide receptor signaling blunts hypertrophy in vitro and in vivo despite sustained pressure load [32,134-136], whereas inhibition of this signaling worsens hypertrophy [137]. Patients with myocardial infarction (MI) leading to cardiomyopathy have poor prognosis despite pharmacological and other treatment modalities [138]. Cell transplantation studies have demonstrated formation of cardiomyocytes and other heart cells from transplanted bone marrow stem cells, cardiac stem cells and ES cells and migration of primitive cells to the heart [139-140]. Although, Islet-1+ (LIM homeodomain transcription factor; Isl1+) cadioblast (endogenous cardiac progenitors that contribute substantially to the embryonic heart) in very low numbers have been identified, and shown to fully differentiate into myocardial lineage [141], it is unlikely these endogenous progenitors will significantly contribute towards the replacement of myocardial cells. In contrast, transplantation of different types of progenitor and stem cells have shown beneficial effects in improving cardiac damage after MI in animal models and many clinical trials [138,142]. A previous study has demonstrated that eNOS in the host myocardium promotes MSC migration to the ischemic myocardium and improves cardiac function through cGMP-dependent increases in SDF-1alpha expression [143]. Similarly, a recent study indicated that early combination of PDE5 inhibitor sildenafil (which prevents the degradation of cGMP) and adipose-derived MSC exhibited greater preservation of left ventricular (LV) function in rat dilated cardiomyopathy model than untreated animals [144] thereby implicating the role of eNOS and cGMP in mediating such effect.

However, most of work has been done with adult stem cells which have limited plasticity. Previous studies demonstrate that transplantation of human ES–derived cardiomyocytes (in coronary ligation model) have shown better improvement of mouse cardiac function up to 4 weeks compared to ES-derived non-cardiomyocytes. However, this effect diminishes after 12 weeks of transplantation [145]. An additional study using ES-derived cardiomycytes indicated that improved mouse cardiac function in a mouse model correlated with vascularity, not the graft size [146]. Although not implicated in the paper, it might suggest the involvement of NO in mediating such effect.

Cardiomycytes derived from human iPS cells also show great potential in regenerative medicine and drug discovery [147]. Unfortunately the effect of NO-cGMP pathway using ES or iPS-derived cardiomyocytes in animal models of cardiac injury has not been reported as of yet. However, based on our in vitro studies and that of other investigators, we propose that NO-cGMP signaling should play a key role in the capacity of stem cell derived cardiomyocytes to engraft, home, and repair damaged myocardium.

Summary and Perspectives

Previous important studies using NOS, sGC and PKG knockout models clearly demonstrate the significant role of NO-cGMP signaling in development. Furthermore, studies conducted by us and others suggest that manipulation of NO-cGMP pathway contributes to cell fate determination of stem cells. Our studies for the first time demonstrated that NO- independent heme-dependent allosteric sGC stimulators such as BAY41 2272 and YC-1 induced differentiation of stem cells. Combination of NO donors and sGC activators further enhanced the differentiation with robust increase in cGMP accumulation. These studies may have strong implications as these mediators can be used as a cocktail to enhance the capacity of stem cell derived cardiomyocytes to improve myocardial, microvascular, regenerative (replacement) and cardiovascular function. However, the effects of the NO-cGMP signaling pathway most likely involve temporal, compartmental/spatial, and localized concentration-dependent autocrine and paracrine responses. Based on our previous studies and those of others, in defining the fundamental role of nitric oxide and cyclic GMP in numerous physiological processes, we suggest that these mediators also participate in stem cell proliferation and differentiation.

Acknowledgments

This work was supported in part by National Institutes of Health (GM076695) and grants from the Dunn foundation, the Welch foundation, Mathers foundation, NASA and the University of Texas. KM would like to thank Balraj Singh and Vivek M Singh for critically reading this review article.

Footnotes

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