1 Department of Molecular, Cellular and Developmental Biology, Yale University
School of Medicine, New Haven, CT 06520, USA
2 Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030,
USA
3 Department of Pediatrics, Yale University School of Medicine, New Haven, CT
06510, USA
4 Department of Pathology, Yale University School of Medicine, New Haven, CT
06510, USA
* Author for correspondence (e-mail: joseph.madri{at}yale.edu)
Accepted 4 February 2004
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SUMMARY |
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Key words: Nitric oxide, Vasculogenesis, Yolk sac, Hyperglycemia, Reactive oxygen species, Mouse
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Introduction |
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Studies with knockout mice have revealed several essential molecules that
participate in vertebrate vasculogenesis, including ephrins, TIE2,
angiopoeitin (ANG), platelet-derived endothelial cell adhesion molecule
(PECAM), vascular endothelial growth factor (VEGF) and basic fibroblast growth
factor (bFGF) (Gerety and Anderson,
2002; Sato et al.,
1995
; Suri et al.,
1996
; Duncan et al.,
1999
; Gerber et al.,
1999
; Yasuda et al.,
1992
). Loss of many of these molecules leads to vascular
dysfunction characterized by enlarged, hyperfused capillaries and embryonic
lethality due to cardiovascular defects. Despite the knowledge gained from
these studies, the regulatory molecules and signaling events underlying vessel
development and patterning remain largely unknown
(Darland and D'Amore, 2001
;
Tallquist et al., 1999
). One
possible candidate is nitric oxide (NO), a small multifunctional gaseous
molecule that acts as a vasoactive agent, signaling molecule and free radical
in mammalian systems (Bogdan,
2001
; Papapetropoulos et al.,
1999
; Schmidt and Walker,
1994
).
NO participates in numerous biological functions that are relevant to
reproduction and embryogenesis, including gene expression, cell growth and
matrix remolding. All nucleated mammalian cells possess at least one of the
three conserved NOS enzymes, neuronal (nNOS), endothelial (eNOS) and inducible
(iNOS), which generate NO by the oxidation of L-arginine. Gouge et al. have
demonstrated that NO was produced by preimplantation murine embryos and that
embryos (two-cell to blastocyst stage) cultured with a NOS inhibitor (L-NMMA)
were developmentally delayed or nonviable
(Gouge et al., 1998). The
authors speculated that NO participates in implantation and demonstrated for
the first time that NO is required for early embryonic development in mice.
Accordingly, eNOS knockout mice display defects in ovulation and oocyte
meiotic maturation, and produce fewer pups
(Jablonka-Shariff and Olson,
1998
).
The importance of NO as a regulator of early developmental events has been
established by studies that demonstrated that the oocyte and preimplantation
embryo are exposed to NO, and oocyte maturation, preimplantation embryogenesis
and implantation require NO (Biswas et al.,
1998; Gagioti et al.,
2000
; Maul et al.,
2003
; Novaro et al.,
1997
; Purcell et al.,
1999
; Sengoku et al.,
2001
; Shukovski and Tsafriri,
1995
; Telfer et al.,
1995
). Moreover, in vitro and in vivo studies showed that
administration of either NOS inhibitors or NO donors hinders development of
preimplantation embryos (i.e. expansion to blastocyst stage), which revealed
the importance of maintaining appropriate levels of NO during preimplantation
embryogenesis (Barroso et al.,
1998
; Biswas et al.,
1998
; Gouge et al.,
1998
; Sengoku et al.,
2001
).
The complex effects of NO are highly dependent on micro-environment, with
several groups reporting diametrically opposing effects of NO, which are
probably due to differences in cell/tissue type, local microenvironment and
redox state (Dulak and Jozkowicz,
2003; Kroncke et al.,
1997
; Shaul,
2002
; Wink and Mitchell,
1998
). The role of NO in the developmental milieu of
vasculogenesis in the murine yolk sac has not been studied. During murine
development, both embryonic and extra-embryonic mesoderm is produced giving
rise to the embryonic and extra-embryonic vasculature, respectively. At E7.5,
extra-embryonic mesodermal cells proliferate forming angioblastic cords
(Palis et al., 1995
). At E8.0,
blood islands fuse establishing the primary capillary network (E8.5), which is
intimately associated with mural cells
(Flamme et al., 1997
;
Risau and Flamme, 1995
;
Pinter et al., 1999
).
Subsequently, by E9.5, the capillary plexus has remodeled into a complex
hierarchy of mature large and small vessels, and a functional vitelline
circulation is established (Folkman and
D'Amore, 1996
).
The yolk sac serves an essential function at the maternal-fetal interface,
and its capacity to tolerate environmental insults is vital for the normal
progression of cardiovascular and embryonic organ development
(Brent et al., 1971;
Freeman et al., 1981
;
Jollie, 1990
;
Lerman et al., 1986
; Waddell
and Marlowe, 1981). Environmental insults during this developmental window
lead to defects in the cardiovascular system, which subsequently affect the
development of several embryonic organs. An in vitro whole embryo culture
model permits study of this crucial developmental period and has shown that
short exposure to hyperglycemia is teratogenic, causing dose dependent growth
retardation, neural tube lesions and yolk sac failure
(Cockroft and Coppola, 1977
;
Pinter et al., 1999
;
Reece et al., 1996
;
Rashbass and Ellington, 1988
;
Sadler, 1980a
;
Sadler, 1980b
;
Freinkel et al., 1986
).
Additionally, this model system mimics the morphological and biochemical
vascular defects in the embryo and yolk sac observed in vivo
(New et al., 1976
;
Mills et al., 1979
;
Sadler and Warner, 1984
).
Though the underlying mechanisms are unknown, the yolk sac has been suggested
as a target of the hyperglycemic insult via excess production of reactive
oxygen species (ROS), and a link between yolk sac injury and multi-system
embryopathy has been proposed (Eriksson
and Borg, 1991
; Eriksson and
Borg, 1993
; Hagay et al.,
1995
; Hunter and Sadler,
1992
; Pinter et al.,
1986
; Reece et al.,
1989
; Reece et al.,
1994
; Wentzel and Eriksson,
1998
; Zusman et al.,
1987
).
In this study, we determined the profile of expression/localization of NOS
isoforms and NO production during the well-defined stages of yolk sac vascular
development. Additionally, we used the in vitro whole conceptus culture system
to describe the stage specific morphological defects induced by temporal
disruption of NO/NOS expression and/or activity induced by NO inhibitors, NO
donors and hyperglycemia. Furthermore, we determined the ability of a NO donor
to rescue vascular defects induced by a known environmental insult:
hyperglycemia. In this study, hyperglycemia was chosen as a
pathophysiologically relevant condition, which has been extensively studied,
known to induce ROS and cause vasculopathy
(Eriksson and Borg, 1991;
Eriksson and Borg, 1993
;
Pinter et al., 1986
;
Wentzel and Eriksson, 1998
).
Finally, we begin to explore the relationships between the hyperglycemia
insult and NO inhibition through a pathway involving ROS.
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Materials and methods |
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Conceptus culture
To study vasculogenesis (E7.5-9.5 in mice) an established in vitro whole
embryo culture was employed (Chen and Hsu,
1982; New, 1978
;
New, 1991
;
Sadler, 1979
). Cultures were
performed as previously described (Pinter
et al., 1999
). Briefly, conceptuses were harvested at 7.5 dpc from
timed-pregnant mice. After complete removal of the trophoblast and Reichert's
membrane, the developmental stage was determined using Downs and Davies'
morphological criteria for staging morphological landmarks of the primitive
streak, neural plate and head fold (Downs
and Davies, 1993
). The conceptuses were cultured in pooled rat
serum for 48 hours and examined under a dissecting microscope for structural
and functional defects (neural tube closure, axial rotation completion, yolk
sac circulation and heart beat). Subsequently, conceptuses were processed for
histological analysis to evaluate complexity of branching, organization,
presence of a hierarchical network of large and small vessels, and vessel
diameter.
The following pharmacological reagents were used: DETA NONOate (half-life of NO release is 20 hours at 37°C/pH 7.4), NG-Monomethyl-L-arginine (L-NMMA), NG-Monomethyl-D-arginine (D-NMMA) (Calbiochem, San Diego, CA) and N?-Nitro-L-arginine methyl ester hydrochloride (L-NAME) (Sigma-Aldrich, St Louis, MO). For the hyperglycemic condition 20 mM D-glucose was used (normoglycemic serum contains 5 mM). Control cultures contained vehicle (PBS), D-NMMA or D-NAME. All reagents were added at the beginning of culture.
Immunohistochemistry and immunofluorescence microscopy
Conceptuses were harvested from timed-pregnant mice or culture conditions
at the indicated time points. The yolk sacs were separated from the embryos
and fixed in 4% PFA. Polyclonal anti-murine PECAM-1
(Pinter et al., 1999) was used
and detected with biotin-conjugated secondary antibody and the ABC kit
(Vector, Burlingame, CA). The reaction was visualized with diaminobenzidine.
For immunofluorescence, after fixation, the samples were flash frozen in
2-methylbutane cooled in liquid nitrogen. Frozen sections of 10 µm were
prepared. Immunodetection was performed using Alexa Fluor 594 (Molecular
Probes, Eugene, OR).
Western blotting
Conceptuses were harvested from timed-pregnant mice or culture conditions
at the indicated time points. Samples of 10-20 were pooled and lysed RIPA
Buffer (Upstate, Waltham, MA) supplemented with Complete Protease Inhibitor
Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktails
(Calbiochem, San Diego, CA). The samples were homogenized and soluble extracts
obtained by centrifugation. Protein concentrations were determined by BCA
Assay (Bio-Rad, Hercules, CA). For western blotting, 40 µg of protein was
loaded onto SDS-PAGE gels under reducing conditions. For immunoprecipitation,
400 µg of lysate was precleared with normal rabbit serum and precipitated
with protein A/G Sepharose. The supernatant was incubated with the primary
antibody, washed with decreased salt conditions and electroblotted. Primary
antibodies used included eNOS, iNOS, Akt and phospho-Akt from Santa Cruz
(Santa Cruz, CA), phospho-eNOS (Ser-1177) (Cell Signaling Technology, Beverly,
MA) and phospho-eNOS (Ser-1177) (Zymed, San Francisco, CA). Luminescence was
preformed using the Western Lightening Chemiluminescence Reagent (PerkinElmer,
Boston, MA). Blots were stripped and reprobed with ERK2 (Santa Cruz, Santa
Cruz, CA) as a loading control.
NO detection
NO localization was performed using
4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate
(Molecular Probes, Eugene, OR), which is the most sensitive reagent currently
available for the detection of low concentrations of NO (detection limit is
3 nM of NO) (Kojima et al.,
1998a
; Kojima et al.,
1998b
; Kojima et al.,
1999
). DAF-FM is cell permeable and nonfluorescent until it
combines with NO to form a fluorescent benzotriazole.
Conceptuses were harvested at 7.5 dpc and incubated with 10 µM DAF-FM for 60 minutes at 37°C. Subsequently, the conceptuses were washed in PBS and incubated for 20 minutes to allow complete de-esterification of intracellular diacetates. After whole mounting the conceptuses, images were immediately captured using an Olympus inverted microscope.
ROS detection
ROS localization was performed using dihydroethidium (DHE), which reacts
principally with superoxide anion. DHE enters the cytoplasm and becomes
oxidized to ethidium, which intercalates into the DNA and produces red
fluorescence. Conceptuses were incubated in the indicated conditions for 24
hours. DHE (10 µM) was added to the culture media for 30 minutes at
37°C. Subsequently, the samples were washed and flash frozen in
2-methylbutane cooled with liquid nitrogen. Slides were immediately prepared
and imaged.
Morphometrics
Yolk sac vessel diameters were measured using the IP Lab Spectrum program.
Micrographs of PECAM1-stained yolk sacs were generated using an Olympus IX71
inverted microscope (20x objective) and saved as Photoshop 5.0 TIFF
files. These files were opened using IP Lab Spectrum and the vessel diameters
of several randomly selected fields measured.
Statistics
The data were analyzed by t-test using StatView (SAS Institute).
P values less than or equal to 0.05 were considered significant.
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Results |
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An inverse relationship between eNOS and iNOS isoforms exists during
vascular development in the yolk sac (Fig.
1). During the blood island formation stage, iNOS protein level is
high, while eNOS protein level is barely expressed. However, a switch in
expression levels of iNOS and eNOS isoforms began just prior to the primary
capillary plexus stage (8.0 dpc) and persisted during the primary plexus
stage. At the vessel maturation stage, iNOS protein was absent while eNOS
protein expression continued. nNOS was not detected in the yolk sac at any of
the three developmental stages examined (data not shown).
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In L-NMMA-treated conceptuses the hierarchy of vessels was lost and capillaries were enlarged while normal vascular development occurred in the presence of D-NMMA and LNMMA + L-Arginine (Fig. 4A-C). Histograms of the size distribution of vessels in the control versus L-NMMA conditions revealed a peak shift from 10-15 µm to 20-25 µm, respectively. The fraction of vessels in the 25-30 to 55-60 µm categories rose from <5% to 5-14% in control versus LNMMA conditions, respectively (Fig. 4F). The median vessel diameter in L-NMMA treated conceptuses harvested at 9.5 dpc was 27.6 µm compared with 30.6 µm in control E8.5 conceptuses (capillary plexus stage), confirming arrest at this stage. Additionally, a functional circulation was not present and the vessels contained fewer blood cells.
In order to determine the developmental window of susceptibility to L-NMMA, streak, neural plate and headfold stage conceptuses were separately treated. L-NMMA induced stage-specific defects in streak and neural plate conceptuses, while headfold stage conceptuses were resistant to the insult. Primitive streak conceptuses displayed the most vulnerability to the insult (i.e. complete arrest at the capillary plexus stage); therefore this stage was used for subsequent studies. Areas of normal vasculature, enlarged vessels and areas devoid of vasculature (endothelial clusters present) were all observed in neural plate stage conceptuses (data not shown).
Exogenous NO treatment results in normal vascular development
Previous studies have shown that high concentrations of NO donor (0.1 and 1
mM DETA/NO) in vivo impaired embryo development while lower concentrations (1
and 10 µM) did not cause developmental abnormalities
(Barroso et al., 1998;
Sengoku et al., 2001
).
Therefore, we supplemented low doses of a slow, steady release NO donor
(NOC-18) to the culture media and assessed the tolerance of the yolk sac to
exogenous NO (20 µM) during vasculogenesis. Whole-mount
immunohistochemistry was performed for PECAM at 9.5 dpc to assess vascular
morphology. Exogenous NO treatment resulted in the formation of a normal
hierarchy of large and small vessels (Fig.
5A,B) and a functional circulation. The size distribution of
vessels in the NOC-18 versus control conditions produced overlapping
histograms (Fig. 5C). Thus, a
dose of 20 µM NOC-18 is not toxic to the vasculature and does not cause
vascular dysfunction. Additionally, the ability of exogenous NO to regulate
the expression of NOS isoforms was evaluated by western blot. Conceptuses were
cultured with 20 µM NOC-18 and harvested at 8.5 dpc, when the switch in NOS
isoforms occurs. Exogenous NO treatment correlated with increased baseline
expression of eNOS and decreased baseline expression of iNOS normally observed
at 8.5 dpc (Fig. 5D).
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|
NO donor rescues the vasculopathy induced by hyperglycemia
To investigate the possibility that NO bioavailability is a mediator of
hyperglycemia-induced vasculopathy, conceptuses were cultured in hyperglycemic
media supplemented with a NO donor (NOC-18). The addition of exogenous NO
resulted in rescue of the vasculopathy induced by hyperglycemia
(Fig. 6B). Morphologically,
normal primary plexus and large vessels formed, establishing a functional
circulation and allowing embryo development to progress. NOC-18 appeared to
restore normal large vessel morphology and branching morphogenesis.
Additionally, vessels were not dilated or enlarged. The size distribution of
vessels in the NOC-18 supplemented hyperglycemic condition resulted in a near
overlapping histogram with the control
(Fig. 6C). Furthermore the
fraction of vessels in the 35-40 to 55-60 µm categories decreased from
5-12% to <7% in the hyperglycemic versus hyperglycemic plus NOC-18
conditions, respectively.
The ability of exogenous NO to restore normal NOS isoform distribution during the hyperglycemic insult was evaluated at 8.5 dpc, when the NOS isoform switch normally occurs. Supplementation of NOC-18 to the hyperglycemic media restored eNOS levels and downregulated iNOS (Fig. 6I).
NO depletion increases ROS production: a common pathway in vasculopathy
Studies using low doses of NO donors demonstrated that NO directly acts as
an antioxidant (Chang et al.,
1996; Joshi et al.,
1999
; Kim et al.,
1995
; Wink et al.,
1993
). Therefore, we hypothesized that the vasculopathy induced by
L-NMMA may be due to increased ROS, suggesting a common pathway with
hyperglycemia. In order to evaluate the potential role of ROS in L-NMMA
induced vasculopathy in the yolk sac, ROS production was evaluated by DHE at
8.0 dpc. This time point, just prior to the primary plexus stage, was chosen
because it represents the point at which the NOS isoforms switch in
distribution (Fig. 1).
Furthermore, NO inhibition seems to cause a defect in primary plexus formation
and arrest at this subsequent stage (8.5 dpc). L-NMMA induced a dramatic
increase in ROS production (Fig.
7B). A gradient of ROS was present from the endoderm to the
mesodermal/endothelial layer, with the highest ROS levels present in the
endoderm. Additionally, cells within the blood islands and endothelial cells
within the vessels expressed ROS. To determine if uncoupling NOSs contributes
to superoxide anion (SO) generation, L-NAME was used. L-NAME, unlike L-NMMA,
inhibits SO production by NOSs. L-NAME treatment induced similar ROS formation
as L-NMMA treatment, suggesting that uncoupling of NOSs does not significantly
contribute to ROS production (Fig.
7C).
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Discussion |
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We next demonstrated that NO depletion arrested vascular development at the
primary plexus stage and induced vasculopathy. The presence of blood islands
suggests that endothelial specification, which begins during gastrulation
(E6.0), and egress of mesodermal cells had occurred. However, as L-NMMA was
added at the primitive streak stage, a time when mesodermal invagination
occurs and mesodermal cell migration is still occurring, these events may be
affected by NO depletion/bioavailability. The subsequent angioblastic cords
were capable of forming a polygonal pattern of capillaries, but the
capillaries were enlarged, typical of the vasculopathy induced in several
diverse knockout mice (Gerety and
Anderson, 2002; Sato et al.,
1995
; Suri et al.,
1996
). These defects have been associated with hyperfusion of
blood islands, which suggests a role for NO in proper blood island formation.
It could also be argued that L-NMMA treatment results in an immature or
arrested vascular network, suggesting a defect in vascular remodeling
processes and a possible role for NO in vessel size determination and vascular
organization. In this case, it is unclear whether the defect is secondary to a
defect in blood island malformation or NO-mediated vascular pruning and smooth
muscle investment events.
Stage-specific defects were associated with L-NMMA, with primitive streak
and neural plate conceptuses showing susceptibility to L-NMMA, while head-fold
stage conceptuses did not. The stage specific defects induced by NO depletion
underscores the influence of the tissue micro-environment on NO-mediated
actions during vascular development. These differences in susceptibility may
be the result of developmental stage specific sensitivity to environmental
conditions (Barroso et al.,
1998; Sengoku et al.,
2001
). The observed sensitivity of primitive streak conceptuses to
changes in NO concentration is currently unexplained; we speculate that
differentiation and migratory events that occur in the splanchnic mesoderm
specifically at this stage may be affected, while in late head fold stage
embryos these events have been completed.
During this stage, blood island formation occurs and iNOS localized to the
visceral endoderm, the major secretory layer that sends inductive signals to
the mesoderm (Antin et al.,
1994; Jollie,
1990
; Wilt,
1965
). The endoderm has been demonstrated to be required for
normal blood island formation, affecting endothelial cell differentiation
(Miura and Wilt, 1969
;
Bielinska et al., 1996
).
Furthermore, endoderm-derived inductive signals were determined to direct
endothelial differentiation during a specific developmental period:
gastrulation (Belaoussoff et al.,
1998
). Molecules such as winged-helix genes, Indian hedgehog and
VEGFA have been discovered as endoderm-derived factors that act locally on the
mesoderm and are required for yolk sac vasculogenesis
(Farrington et al., 1997
;
Dyer et al., 2001
;
Damert et al., 2002
). Given
the effects of NO depletion during late gastrulation/early organogenesis on
vascular formation, we speculate that iNOS derived NO is one of the
endoderm-derived molecules that acts as a paracrine regulator of endothelium
development via direct or indirect effects (NO-mediated production of
essential factors).
To evaluate the effect of excess NO on vascular development, we applied a
low dose of NO donor to the culture media and found that NOC18 did not cause
vascular defects. Furthermore, NOC18 treatment correlated with increased eNOS
and decreased iNOS during primary capillary plexus formation. At this time we
can not confirm that the induction of eNOS is a direct or indirect effect of
NO. Potentially, feedback loops exist in which NO regulates endodermal growth
factors that subsequently affect NOS expression. For example, NO is known to
induce VEGF, which has been demonstrated to increase eNOS
(Cha et al., 2001;
Dulak and Jozkowicz, 2003
).
Alternatively, the conceptus may be capable of NO sensing and modulation of
NOS isoforms by yet unidentified mechanisms, in order to maintain the
appropriate concentration of NO during organogenesis. This switch between NOS
isoforms just prior to the primary plexus stage seems to represent an
important event in the progression of normal vascular development. Its
significance is further demonstrated by the hyperglycemic experiments.
Hyperglycemia resulted in the persistence of the iNOS isoform and depression of the eNOS isoform. Potentially, in order for normal primary plexus formation to occur, NO-dependent signaling mechanisms are required in the endothelium. Therefore, the switch from endodermal NO production to endothelial NO production and autocrine NO-mediated events must occur. We speculate that the abrupt decline in iNOS represents a phase of normal development when a sustained increase in NO production via iNOS may alter further vascularization.
Though hyperglycemia increased endodermal NO production at 7.5 dpc, we
observed that NO donor treatment rescued the hyperglycemia-induced
vasculopathy and restored the eNOS/iNOS distribution. This seemingly
conflicting result may be explained by decreased bioavailability of NO leading
to a requirement for additional NO. Excessive NO produced by the high NO
output NOS isoform, iNOS, is upregulated in vascular diseases and known to
cause cytotoxicity and vascular dysfunction
(Hibbs et al., 1989;
Moncada et al., 1991
;
Zhang et al., 2003
). However,
despite increased production of NO in vascular diseases, there is impaired
vasodilation, suggesting decreased NO bioavailability
(Minor et al., 1990
).
It has been demonstrated that glucose scavenges NO in a dose-dependent
manner by the formation of a glucose-NO adduct, providing evidence for a
direct action of glucose on NO bioavailability
(Brodsky et al., 2001).
Additionally, hyperglycemia increases the production of ROS, of which SO is
known to combine with NO and deplete it from the system, thus inhibiting NO
mediated intracellular signaling and downstream cellular responses
(Mügge et al., 1991
;
Urbich et al., 2002
;
Zhang et al., 2001
). The
reaction between SO and NO is rapid, occurring at a rate of
6.7x109 M-1s-1, a near
diffusion-limited rate (Huie and Padmajas,
1993
). This reaction occurs about six times faster than the
dismutase of SO by superoxide dismutase (SOD), thus NO is an efficient
scavenger of SO (Koppenol,
1998
). By increasing the concentration of NO, once saturation of
SO has occurred, the excess NO is available to act in the endoderm.
Furthermore, NO is membrane permeable, unlike SO, and capable of diffusing
over 100 µm in a few seconds at 37°C
(Meulemans, 1994
;
Wise and Houghton, 1969
;
Malinski et al., 1993a
;
Malinski et al., 1993b
). NO
has been demonstrated to diffuse through tissues without consumption,
establishing its role as an intracellular messenger
(Lancaster, 1994
;
Wood and Garthwaite, 1994
).
Therefore, it is possible for NO released by the donor to diffuse through the
single layer of endodermal cells and reach the mesoderm, where SO
concentrations are lower.
The diffusion capabilities of the NO donor and its ability to restore the
normal eNOS/iNOS distribution in the hyperglycemic condition may provide the
required local concentrations of NO for vascular development to progress
normally. As eNOS produces a lower concentration of NO than does the iNOS
isoform, we expect the physiological concentration of NO during normal
development in the endothelium to be low. In other tissues, such as the
cerebrum, the concentration of NO was measured at 10 nM during physiological
conditions (Malinski et al.,
1993a; Malinski et al.,
1993b
). Furthermore, only 5 nM of NO is needed to induce
relaxation of vessels and is the minimum concentration to activate guanylate
cyclase. Thus, low concentrations of NO are sufficient for NO-mediated actions
in physiological conditions and, potentially, developmental states. We
demonstrated that, under normal conditions, a small fraction of eNOS was
phosphorylated; thus, we speculate that eNOS produces a low [NO] which is
required for optimal vascular development. The survival factor, Akt, is known
to activate eNOS (Fulton et al.,
1999
; Michell et al.,
1999
). Furthermore, Akt plays a role in endothelial cell survival
as blood vessels assemble during vascular development through a mechanism
involving RhoB (Adini et al.,
2003
). As phospho-Akt was detected in E8.5 yolk sacs, this pathway
may serve as a mechanism to activate eNOS and also promote endothelial cell
survival.
The protective effect of upregulation of the low NO output NOS isoform,
eNOS, by the NO donor during the hyperglycemic insult is supported by several
studies in eNOS knockout mice, which revealed that this isoform serves
cardioprotective and vasoprotective functions
(Connelly et al., 2003;
Gewaltig and Kojda, 2002
;
Jablonka-Shariff and Olson,
1998
). These functions include modulation of vasoactivity and
blood pressure, and inhibition of platelet aggregation, leukocyte adhesion and
smooth muscle cell proliferation (Limbourg
et al., 2002
; Jugdutt,
2002
; Papapetropoulos et al.,
1999
). Additionally, several studies have demonstrated that NO
acts as an antioxidant (Chang et al.,
1996
; Hermann et al.,
1997
; Joshi et al.,
1999
; Kim et al.,
1995
; Paxinou et al.,
2001
; Wink et al.,
1993
). Accordingly, we showed that NO supplementation to
hyperglycemic media reduces hyperglycemia-induced ROS. Furthermore, we
demonstrated that L-NMMA treatment induced ROS in the endoderm and
mesoderm/endothelium. These results suggest that endogenous NO has antioxidant
functions during blood island formation.
The expression pattern of ROS in L-NMMA treated conceptuses was similar to
that induced by hyperglycemia, indicating that the induced vasculopathies may
share a common pathway of ROS induced vascular defects. High concentrations of
ROS injure tissues and cause vascular dysfunction
(Channon and Guzik, 2002).
Accordingly, mice deficient in copper-zinc superoxide dismutase, the
cytosol/nuclear SOD, display increased SO, decreased relaxation response to
NO, and vascular dysfunction (Didion et
al., 2002
). Furthermore, administration of superoxide scavengers
protects against vascular dysfunction in experimental animal models
(Iadecola et al., 1999
;
Mayhan, 1997
).
Under normal conditions in vivo, ROS and NO levels are balanced and low,
and thus their combination is limited by their ability to diffuse and interact
with each other. In this situation, both ROS and NO are essentially free to
initiate their respective signaling cascades. NO has been demonstrated to
interact with several intracellular signaling cascades, including
mitogen-activated protein kinase (MAPK), janus kinase (JAK) and Jun N-terminal
kinase (JNK) (Lander, 1997;
Kim et al., 1997
;
So et al., 1998
).
Additionally, transcription of several gene classes, including cytokines,
matrix proteins and hormones, are modulated by NO regulation of nuclear factor
B (NF
B), hypoxia inducible factor 1 (HIF1) and zinc-finger
transcription factors (Huang et al.,
1999b
; Kroncke and Carlberg,
2000
; Matthews et al.,
1996
; Tabuchi et al.,
1996
; Torres and Forman,
2000
). ROS, however, regulates transcription of protein kinases,
growth factors and transcription factors, and plays a role in signaling in the
vasculature (Burdon, 1996
;
Giaccia and Kastan, 1998
;
Maulik, 2002
;
Meyer et al., 1994
;
Nose, 2000
). Additionally, ROS
inhibits low molecular weight (LMW) phosphatases, which have been shown to
downregulate proliferation by attenuating growth factor receptor signaling
(Chiarugi, 2001
;
Huang et al., 1999a
). It is
essential for ROS and NO to carry out these respective signaling cascades
because, if they interact, as they do at high concentrations, vasculopathy
occurs.
Taken together, the results of this study suggest that tight regulation of
the stage appropriate levels of NO and NO-mediated events are required for
normal vascular development. The downstream NO-mediated signaling events and
molecules are yet to be determined, as is the crosstalk between NO and other
endoderm-derived factors (Fig.
8). However, the observed antioxidant effect of NO during early
vasculogenesis broadens the protective activities of the yolk sac endodermal
layer. Future studies using this model are needed to examine the role of NO in
antioxidant events during vasculogenesis, particularly the regulation of SODs
and other protective mechanisms such as peroxisome proliferator activated
receptor , PPAR
. Moreover, the relationship between
high glucose, NO bioavailability and downstream antioxidant events needs to be
further explored.
|
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ACKNOWLEDGMENTS |
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