First published online May 5, 2004
doi: 10.1242/10.1242/dev.01131
Development 131, 2485-2496 (2004)
Published by The Company of Biologists 2004
Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy
Anjali K. Nath1,
Josephine Enciso2,
Misako Kuniyasu3,
Xiao-Ying Hao3,
Joseph A. Madri4,* and
Emese Pinter3
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
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Fig. 1. Temporal distribution of endogenous NOS isoforms during vascular
development. (A) ERK2 normalized data for NOS protein expression in pooled in
vivo grown E7.5, E8.5 and E9.5 yolk sacs. The broken line represents eNOS,
whereas the unbroken black line represents iNOS (n=4,
*P<0.05, **P<0.01). The
quantified signals represent mean±s.e.m. The relative expression levels
were compared with 7.5 dpc levels. (B) Representative western blot of the
graphed data.
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Fig. 2. 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 with the blood island
formation, primary capillary plexus and vessel maturation stages,
respectively. Immunofluorescence for eNOS (A-C) and iNOS (D-F) was performed
(red, NOS; blue, DAPI). Images of single blood islands or vessels stained with
NOS were captured at 40x magnification and merged with DAPI images. En,
endoderm; Me, mesoderm/endothelium (n 4).
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Fig. 3. Production of NO in 7.5 dpc conceptuses. NO production and localization
were preformed using DAF-FM. (A) Phase-contrast image of a 7.5 dpc conceptus
showing the intact mesoderm (outlined by the broken red line) and the removed
endoderm. (B) DAF-FM fluorescence (green) of the corresponding area. (C)
Phase-contrast image of an enlargement of the boxed area in A containing the
endodermal layer. (D) DAF-FM fluorescence of the corresponding area. (E)
Representative western blots of eNOS and p-eNOS from eNOS immunoprecipitates
of 8.5 dpc yolk sac lysates. (F) Representative western blots of Akt and p-Akt
from 8.5 dpc yolk sacs.
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Fig. 4. L-NMMA inhibits yolk sac vascularization. The vascular morphology of
L-NMMA-treated conceptuses was evaluated by PECAM1 staining at 9.5 dpc: (A)
D-NMMA, (B) L-NMMA and (C) L-NMMA plus L-arginine. Hematoxylin and Eosin
stained images of yolk sacs treated with D-NMMA (D) and L-NMMA (E). (F)
Morphometric analysis of images from PECAM1-stained yolk sacs showing vessel
diameter distribution. Experiments were repeated at least five times.
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Fig. 5. NO Donor effects on vascular morphology and NOS distribution. The vascular
morphology of NOC18-treated conceptuses was evaluated by PECAM1 staining at
9.5 dpc: (A) control and (B) NOC18. Morphometric analysis of images from
PECAM1-stained yolk sacs showing vessel diameter distribution (C).
Representative western blot of the effect of a NO donor on NOS protein
distribution at 8.5 dpc (D). Experiments were repeated at least five
times.
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Fig. 6. Effects of hyperglycemia on iNOS/eNOS distribution and NO production, and
rescue by NO donor. The ability of NOC18 to restore normal vascular morphology
of hyperglycemic treated conceptuses was evaluated by PECAM1 staining at 9.5
dpc: (A) hyperglycemia and (B) hyperglycemia plus NOC18. Morphometric analysis
of images from PECAM1-stained yolk sacs showing vessel diameter distribution
(C). Graphs of ERK2 normalized data for iNOS (D) and eNOS (E) protein
expression in pooled 7.5, 8.5 and 9.5 dpc conceptuses. The broken lines
represent the hyperglycemic condition, whereas the unbroken black lines
represent the control (n=4, *P<0.05,
**P<0.01). The data (mean±s.e.m.) is relative to
the control 7.5 dpc levels. Above each graph is a representative western blot
of iNOS and eNOS at 8.5 dpc. (F) Phase-contrast image of the endoderm of a 7.5
dpc conceptus showing the intact endodermal surface. (G) DAF-FM fluorescence
of the corresponding area of a conceptus cultured in normoglycemic conditions.
(H) DAFFM fluorescence of a similar area of a conceptus cultured in
hyperglycemic conditions. (I) Representative western blot of the effect of a
NO donor on NOS protein distribution of glucose treated conceptuses at 8.5 dpc
and graph of ERK2 normalized averaged data for eNOS and iNOS protein
expression (white columns represent eNOS and black columns represent iNOS;
averages of two experiments). Nml, normoglycemic; HG, hyperglycaemic; HG +
NOD, hyperglycemic + NOC-18.
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Fig. 7. Induction of ROS: a common pathway in vasculopathy. ROS production and
localization in the yolk sac was evaluated by DHE (red) in 8.0 dpc conceptuses
treated with D-NMMA (A), L-NMMA (B), L-NAME (C), glucose (D) and glucose plus
NOC-18 (E). Images were captured at 20x magnification and merged with
DAPI images (n=4).
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Fig. 8. Working model of the role of NO and the teratogenic compounds high glucose
and L-NMMA during the development of the yolk sac vasculature in the murine
conceptus. During normal development, stage-specific production of NO by
endodermal iNOS elicits autocrine responses in endodermal cells,
downregulating ROS production and paracrine responses in endothelial cells,
and upregulating eNOS expression. These autocrine and paracrine actions of NO
ultimately facilitate vascular differentiation and development. In the
hyperglycemic condition, endodermal iNOS expression is maintained and the
resultant increased NO generated participates with SO to generate increased
ROS levels, which, in turn, affect the numerous soluble factors required for
mesodermal differentiation and migration. In the presence of L-NMMA,
developmental stage-specific iNOS production of endodermal NO is abrogated,
resulting in the blunting of NO autocrine and paracrine signaling, which, in
turn, affects the myriad of soluble factors required for mesodermal
differentiation and migration. In the presence of a NO donor,
glucose-inhibited yolk sac vascularization proceeds. The exogenous NO
re-establishes the normal timing of the switch in eNOS/iNOS levels and
reconstitutes the NO-driven endodermal and mesodermal signaling pathways,
facilitating normal vascular differentiation and development.
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© The Company of Biologists Ltd 2004
