First published online 19 November 2003
doi: 10.1242/dev.00887
Development 130, 6465-6474 (2003)
Published by The Company of Biologists 2003
Retinoic acid regulates endothelial cell proliferation during vasculogenesis
Lihua Lai1,2,3,*,
Brenda L. Bohnsack2,3,4,*,
Karen Niederreither3,4,5,
and
Karen K. Hirschi1,2,3,4,6,,
1 Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030,
USA
2 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX
77030, USA
3 Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX
77030, USA
4 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA
5 Department of Medicine, Baylor College of Medicine, Houston, TX 77030,
USA
6 Center for Cardiovascular Development, Baylor College of Medicine, Houston, TX
77030, USA
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Fig. 2. Endothelial cell differentiation and maturation. Whole-mount immunostaining
with antibodies against PE-CAM1 demonstrated that in wild-type yolk sacs (A),
endothelial tubes were similar in diameter, evenly distributed and exhibited
some `pruning' into smaller vessels (arrows). Endothelial tubes within
Raldh2-/- yolk sacs (B) were dilated (arrows), with no
appropriate remodeling. Within wild-type embryos, vascular plexus formation
was evident within the head (C) with distinct margins of vascularization
(arrows); vascular plexi within the heads of Raldh2-/-
embryos (D) were diffusely distributed. Vessels within the somitic regions of
Raldh2-/- mutants were also dilated and inappropriately
patterned (F, arrows) relative to wild type (E). Semi-quantitative RT-PCR
analyses revealed that the expression of Flk1, VE-cad, Tie2 and Ang1 mRNA in
Raldh2-/- mutants was similar to wild type, but the
expression of Cx40 mRNA in Raldh2-/- mutants was
significantly reduced (G).
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Fig. 3. Control of endothelial cell proliferation. Embryos and yolk sacs were
dissected at E8.25-8.5, and tissues were immunostained with antibodies against
phosphohistone 3. Compared with wild type (A), there was consistently more
phosphohistone 3 staining in Raldh2-/- tissues (B). The
mitotic index was significantly higher in Raldh2-/-
tissues compared with wild type at E8.5 (6.3±1.5% versus
2.7±1.0%, respectively). Immunostaining of yolk sac cross-sections with
phosphohistone 3, and co-staining with DAPI, revealed no difference in
cellular mitosis in the endodermal layer of yolk sac, but a significantly
higher mitotic index within the mesodermal layer of
Raldh2-/- yolk sacs, compared with wild type (C).
Coimmunostaining for phosphohistone 3 and VE-cadherin indicated that the
proliferating mesodermal cells in Raldh2-/- yolk sacs were
predominantly endothelial cells (E); proliferating endothelial cells in
wild-type yolk sac were infrequently observed (F).
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Fig. 4. RA regulation of p21 in vivo and in vitro. Whole-mount staining with
antibodies against p21 revealed significantly reduced levels of p21 in
Raldh2-/- yolk sacs (B) compared with wild type (A).
Section staining revealed p21 expression predominantly in endothelial cells in
wild-type yolk sacs (C, arrows); p21 was essentially absent in
Raldh2-/- yolk sac (D). Endothelial cells were cultured
for up to 72 hours in the presence or absence of 1 µM RA. Western analyses
revealed that RA induced the expression of p21 in endothelial cells within 8
hours (E).
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Fig. 5. Retinoic acid regulation of endothelial cell cycle progression. (A)
Endothelial cells were cultured (10,000/well) for 72 hours in control
conditions or with 0.5 µM RA, 0.5 µM MA or 0.5 µM TTNPB. RA
significantly suppressed the increase in endothelial cell number seen over
time in culture in control cells, in a process specifically mediated via RAR
receptors (A). (B) Fixed control and RA-treated endothelial cells were stained
with propidium iodide and subjected to FACS. RA significantly decreased the
proportion of endothelial cells in S phase, and increased the proportion of
cells in G1 phase, but did not induce apoptosis.
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Fig. 6. Mechanism by retinoic acid regulates endothelial cell cycle progression.
Total protein was isolated from control or RA-treated endothelial cells,
immunoprecipitated with antibodies against Cdk4, p21 or p27, and subjected to
western analyses. Complex formation between cyclin D1 or D2 and Cdk4 was
significantly reduced in RA-treated endothelial cells, whereas complex
formation between RA-induced p21 and Cdk4, as well as cyclins D1 and D2, was
greatly enhanced (A). There were lower levels of phosphorylated Rb protein in
RA-treated endothelial cells, compared with controls (B).
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Fig. 7. Circulating RA rescues Raldh2-/- vascular defects.
Pregnant Raldh2+/- females were fed chow containing 0.1
mg/g all-trans RA from E7.5-8.5, E7.5-9.5 or E7.5-12.5, killed at E12.5 and
examined histologically. Mutants were compared with wild-type embryos from
mothers fed RA from E7.5-12.5 (first column), which were no different from
wild-type embryos from chow-fed mothers. In mutants exposed to RA from
E7.5-8.5 (second column), yolk sacs exhibited formation of some large vessels
(arrowheads), but all smaller caliber vessels (arrows) were significantly
dilated. Exposure to RA from E7.5-9.5 (third column) induced formation of a
larger number of branched vessels (arrowheads), but dilation of small vessels
(arrows) was still evident. Exposure to RA from E7.5-12.5 (fourth column),
lead to vascularization of mutant yolk sac equivalent to wild type; large
(arrowheads) and small (arrows) vessels were similar in caliber and
distribution. The mitotic index of yolk sac tissue (bottom row) from `rescued'
mutants was evaluated via immunostaining for phosphohistone 3 and
significantly elevated for all, compared with wild type, except those exposed
to RA at E7.5-12.5.
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© The Company of Biologists Ltd 2003
-actin
expression was evident around large vessels. In Raldh2-/-
yolk sacs (H), investment of endothelial tubes by SM-