Department of Physiology, University of Arizona, Tucson, AZ 85724, USA
* Author for correspondence (e-mail: amsimon{at}u.arizona.edu)
Accepted 13 February 2003
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Summary |
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Key words: Connexin37, Connexin40, Gap junction, Intercellular communication, Aorta, Endothelium
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Introduction |
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Nakamura et al., 1999;
van Kempen and Jongsma, 1999
;
Cai et al., 2001
;
Haefliger et al., 2001
;
Rummery et al., 2002
). In
addition, Cx43 is found in a subset of endothelial cells in the rat arterial
vasculature (Gabriels and Paul,
1998
).
Vascular gap junctions could serve several physiological roles. Coupling of
vascular endothelial cells is thought to facilitate conduction of vasomotor
responses along arterioles by allowing for cell-to-cell transfer of electrical
signals (Segal and Duling,
1989; Emerson and Segal,
2000
). Indeed, Cx40/ mice exhibit
diminished conduction of arteriolar dilatation in response to acetylcholine
and bradykinin, and they are hypertensive
(de Wit et al., 2000
). In
conduit vessels, gap junctions have been implicated in a role in regulating
vascular tone (Christ et al.,
1996
; Chaytor et al.,
1998
). Junctional communication may also be important in the
control of vascular cell proliferation and migration
(Larson and Haudenschild,
1988
; Pepper et al.,
1989
; Kurjiaka et al.,
1998
). In this regard, gap junctions could have important roles in
the development of the vasculature and in responses to blood vessel wounding.
Despite significant knowledge of connexin expression patterns in blood
vessels, the contribution of specific connexins to endothelial communication
is still not well understood. In particular, the relative contributions of
Cx37 and Cx40 to endothelial coupling has not been fully investigated. While
this work was in progress, Krüger et al. investigated the effects of a
deficiency of Cx40 and concluded that intercellular transfer of injected dyes
was altered in Cx40-deficient aortic endothelium and that expression of
endothelial Cx37 was upregulated
(Krüger et al., 2002
). In
this report, we used intercellular dye-transfer to assess interendothelial
communication in wild-type, Cx37/,
Cx40/,
Cx37+/Cx40/ and
Cx37/Cx40/ aortic segments
isolated from mice of different age groups. In addition, we performed western
blots and RNA analysis to determine whether connexin deletion alters the
levels of non-ablated connexins. We found that Cx37 and Cx40 are collectively
crucial for endothelial communication in mouse arteries and are mutually
dependent on each other for optimal expression in vascular endothelium.
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Materials and Methods |
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Antibodies
Anti-Cx37, anti-Cx40 and anti-Cx43 sera were provided by David Paul
(Harvard Medical School, MA) and were affinity purified
(Beyer et al., 1987;
Gabriels and Paul, 1998
).
Selected experiments were confirmed with a commercial Cx37 antibody (Cx37
A11-A) from Alpha Diagnostic International (San Antonio, TX). The following
antibodies were obtained commercially: anti-VE-cadherin (11D4.1), from BD
Biosciences-Pharmingen (San Diego, CA); anti-caveolin-1, from BD
Biosciences-Transduction Laboratories (Lexington, KY);
anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1) (M-20) for
western blots, from Santa Cruz Biotechnology (Santa Cruz, CA); anti-PECAM-1
(MEC 13.3) for immunostaining, from BD Biosciences-Pharmingen; and
anti-smooth-muscle actin (1A4), from Sigma-Aldrich (St Louis, MO).
Western blotting
Alkaline-extracted endothelial membranes were collected by passing 200
µl of lysis solution (20 mM NaOH and protease inhibitors) three times
through the lumen of thoracic aortas, using a 22-24 gauge needle. Only the
lumen of the aorta was exposed to lysis solution. Lysates collected from six
aortas were pooled. Equal lengths of aorta were extracted in each group.
Lysates were passed through a 26-gauge needle ten times and a sample was
removed for protein determination. Lysates were spun at 100,000
g for 40 minutes to pellet membranes, which were resuspended
in 30 µl of SDS sample buffer. Whole aorta samples (five aortas per group)
were collected by removing fat and adventitia and mincing the vessels in 5 mM
tris-HCl (pH 7.4), 5 mM EDTA, 5 mM EGTA. Samples were Dounce homogenized (40
strokes) and NaOH was added to a final concentration of 20 mM. After 30
minutes on ice, membranes were pelleted and resuspended in 100 µl of SDS
sample buffer. Medial-layer-only samples (five aortas per group) were
collected by removing fat and adventitia and extracting the endothelium once
with SDS sample buffer (100 µl per 5 aortas) followed by lumenal rubbing
with a tungsten wire to remove any residual endothelium. Immunostaining
control experiments showed that the endothelium was efficiently removed.
SDS-extracted aortas were rinsed well with PBS and then processed as described
for whole aortas. Samples were boiled for 5 minutes and run on a 12%
SDS-polyacrylamide gel. Cx37, Cx40, Cx43, caveolin-1, VE-cadherin, and PECAM
were detected on the same nitrocellulose blots. Following primary antibody
incubations, membranes were incubated with horse radish peroxidase
(HRP)-conjugated secondary antibodies (Pierce Endogen, Rockford, IL) and then
processed for chemiluminescence. VE-cadherin blots were incubated with
biotinylated secondary antibody and ABC reagent (Vector Laboratories,
Burlingame, CA). Bands were quantified from film by densitometry using a
BioRad imaging system. Cx37, Cx40, VE-cadherin and caveolin-1 levels were
normalized to PECAM signals. Signals obtained from knockout samples were
expressed as a percentage of wild-type signals.
Immunohistochemistry
Tissues were frozen unfixed in Tissue-Tek OCT embedding medium (Sakura
Finetek, Torrance, CA) and sectioned at 10 µm. Sections were fixed in
acetone at 20°C for 5 minutes, blocked in a solution containing
PBS, 4% fish skin gelatin, 1% goat serum, 0.25% Triton X-100, and incubated
with primary antibodies. Sections were washed, incubated with CY3-conjugated
secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA),
washed and viewed with an Olympus BX51 fluorescence microscope. Images were
captured with a Photometrics SenSys 1401 CCD camera. For en face
immunostaining, aortas were fixed by perfusion with 0.5% paraformaldehyde and
cut open before blocking and incubating with connexin antibodies.
RT-PCR
RT-PCR was performed with RNA obtained from three groups each of wild-type
mice, Cx37/ mice and Cx40/
mice. Each group consisted of six animals (6-7 weeks old). RNA was isolated
from thoracic aorta endothelium by passing 200 µl of lysis solution (4 M
guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sodium lauryl sarcosinate,
0.1 M ß-mercaptoethanol) once through the lumen using a 22-gauge needle.
Lysates from six aortas were pooled and passed through a 26-gauge needle ten
times. Total RNA was isolated according to Chomczynski and Sacchi
(Chomczynski and Sacchi, 1987).
RNA was isolated from whole aortas by removing fat and adventitia and
homogenizing the aortas in lysis solution. RNA was treated with DNAse I and
resuspended in 30 µl. RNA, 6 µl, was reverse-transcribed using an oligo
dT18 primer. The final volume was adjusted to 100 µl. 5 µl of
cDNAs were used for PCR in 100 µl reactions using Advantage 2 polymerase
(BD Biosciences-Clontech, Palo Alto, CA). After 3 minutes at 94°C, five
cycles of touch-down PCR were performed with a 1°C decrement in annealing
temperature after each cycle. The initial cycling parameters were: 94°C
denaturation (30 seconds), 69°C annealing (30 seconds), 72°C extension
(30 seconds). Cycles 6-33 were performed with the following parameters:
94°C denaturation (30 seconds), 64°C annealing (30 seconds), 72°C
extension (30 seconds). 20 µl samples were removed at cycles 25, 27, 29, 31
and 33. Cx37, Cx40, Cx43 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
bands were quantified. Connexin signals were normalized to GAPDH levels.
Signals obtained from knockout samples during the linear portion (cycle 29 was
used) were expressed as a percentage of the signal obtained from wild-type
samples. The primers used for PCR were as follows:
Dye-transfer experiments
Postnatal thoracic aortas were cut into three segments. Embryonic aortas
were maintained as one piece. Segments were pinned out, stained briefly with
25 µM Hoechst 33342 (Molecular Probes, Eugene, OR) to visualize nuclei,
then submerged in PBS containing 0.90 mM Ca2+, 0.49 mM
Mg2+ and 1% BSA during microinjections. Two types of tracers were
used: (1) a mixture of 5% biocytin (Mr 372, neutral charge) and 10
mg/ml dextran-fluorescein (Mr 3x103) (both
from Molecular Probes); or (2) 5 mM
[2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium
(Mr 266, +1 charge), a fluorescent compound referred to as NBD-TMA
(provided by Stephen Wright, University of Arizona, AZ)
(Bednarczyk et al., 2000
).
Microelectrodes were positioned at the surface of an endothelial cell in the
region of the nucleus. Tracer was injected for 10 seconds using the
capacitance overcompensation feature of the amplifier. For NBD-TMA,
dye-transfer was for one minute before counting labeled cells. Transfer of
biocytin was for 15 minutes before the segments were fixed in 4%
paraformaldehyde. Segments were washed and blocked in PBS containing 2% BSA
and 0.25% TX100. Vessels were incubated in tetramethylrhodamine
(TMR)-NeutrAvidin (Molecular Probes), washed, mounted on slides in PBS, and
the number of biocytin-labeled cells counted.
Silver nitrate staining
Silver nitrate staining of aortic endothelial cells was performed
essentially as described by McDonald et al.
(McDonald, 1994). This
technique stains material in the intercellular space, outlining endothelial
cells and marking gaps between cells.
Survival study
Sixteen wild-type and 16
Cx37+/Cx40/ mice (both
129/Sv-C57BL/6 strain) were housed under identical conditions. Animals were
checked daily for 2 years. The percentage of surviving animals was plotted at
two-week intervals.
Statistics
Data were compared statistically using ANOVA (dye injections) or t-test
(westerns and RT-PCR).
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Results |
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Surprisingly, a significant decrease in non-ablated connexins was observed
in aortic endothelium of Cx37/ and
Cx40/ mice (Fig.
1A) (Table 1). The
biggest decline occurred in the levels of Cx37 present in
Cx40/ aorta, where a 17-fold drop was observed on
blots of aortas from 3-7-week-old Cx40/ mice.
Likewise, a 4.2-fold decrease in Cx40 was shown in aortic endothelium from
Cx37/ mice of the same age. In contrast to the drop
in connexin levels, VE-cadherin levels were not reduced on the same blots of
Cx37/ and Cx40/ aorta, nor
was there a change in PECAM levels (Fig.
1A) (Table 1).
Thus, the low levels of non-ablated connexins in the knockout mice were not
due to a generalized decrease in endothelial membrane proteins. We also probed
the blots with an antibody against caveolin-1, a protein that is highly
expressed by vascular endothelial cells and which was recently shown to
interact with some connexins (Schubert et
al., 2002) (Fig.
1A) (Table 1).
Caveolin-1 levels were slightly decreased in both
Cx37/ and Cx40/ samples from
3-7-week-old mice. To determine whether changes in connexin levels were
persistent over time, we performed western blots on aortic endothelium from
4-8-month-old mice (Fig. 1A)
(Table 1). In these animals,
Cx37 protein was 3.0-fold lower in Cx40/ aortic
endothelium compared with wild-type controls, and Cx40 was 2.6-fold lower in
Cx37/ endothelium. VE-cadherin levels were not
significantly altered in the knockout samples, although there was considerable
variability in the VE cadherin measurements from aortas of older animals.
Caveolin-1 and PECAM were not changed in aortic endothelium from 4-8-month-old
Cx37/ and Cx40/ mice. These
results indicate that alterations in the levels of non-ablated connexins in
Cx37/ and Cx40/ aortic
endothelium are long lasting, although in the case of Cx37, the reduction was
more pronounced in younger animals than older animals.
Western blots were also done to determine whether there were changes in
Cx37 in whole aorta, or medial-layer-only fractions of aorta, from
6-7-week-old Cx40/ mice
(Fig. 2A,B). Instead of a
decrease in Cx37, a blot of alkaline-extracted membranes from whole aorta
showed a slight increase (1.4-fold) in Cx37 in
Cx40/ aorta, after normalizing to PECAM
(Fig. 2A). On the same blot,
Cx43 was elevated
fourfold in Cx40/ aorta
(Fig. 2A). The fact that
endothelial Cx37 was diminished in aortic endothelium but not in whole aorta
from Cx40/ animals suggested that Cx37 might be
elevated in the medial layer of Cx40/ aortas. To test
this idea, aortic endothelium was extracted with SDS sample buffer and the
endothelium-free medial layer was homogenized. Alkaline-treated membranes were
collected from the medial layer homogenate and analyzed by western blotting
(Fig. 2B). Cx37, but not Cx40,
was detected in the medial layer samples and the Cx37 signal increased
4.4-fold in the Cx40/ versus wild-type media. The
increase in medial Cx37 paralleled a 4.0-fold increase in Cx43 that was
observed with the same samples. The SDS sample buffer lysates of endothelium
were also analyzed and Cx37 was found to be reduced by 8.2-fold in
Cx40/ endothelium
(Fig. 2B). Similar results for
both endothelial and medial layer Cx37 were obtained using the alternative
Alpha Diagnostics Cx37 antibody (not shown). Blots for Cx40, Cx43 and PECAM
showed little, if any, contamination of endothelial and medial-layer fractions
(Fig. 2B).
Immunofluorescent staining was performed to confirm the changes in connexin levels observed by western blotting. Frozen sections of thoracic aorta from 7-week-old wild-type, Cx37/ and Cx40/ mice were incubated with anti-connexin antibodies (Fig. 3A-P, Fig. 4A-D). Cx37 was predominantly detected in the endothelium of wild-type aorta. At high magnification, however, faint punctate signals in the medial layer could also be observed following Cx37 immunostaining (Fig. 4A,E). Medial layer staining was likewise detected on sections of 4-month-old wild-type aorta. Cx37 immunostaining has not previously been reported in the medial layer of mouse aorta, perhaps because of the relatively low signal in the media compared with the strong signal in endothelium. Therefore, to determine whether the faint fluorescent signals in the medial layer were genuinely due to the presence of Cx37 and not due to antibody cross reactivity, we compared Cx37 immunostaining in wild-type and Cx37/ aorta (Fig. 4A,B). Both the strong endothelial staining and the weak medial staining were absent in Cx37/ aorta sections, indicating that the medial layer staining was specific for Cx37. Next, we immunostained wild-type and knockout aorta sections to look for changes in non-ablated connexins (Fig. 3A-P, Fig. 4). Consistent with the western blot results, we observed a prominent drop in the endothelial signal of Cx37 in sections of Cx40/ aorta. Only occasional punctae were observed in the endothelial cell layer of Cx40/ aorta. By contrast, Cx37 appeared elevated in the medial layer of Cx40/ aorta, as the faint Cx37 signal in this layer was easier to detect in the Cx40/ sections than in wild-type sections (Fig. 3K, Fig. 4C). Aortas from 4-month-old Cx40/ animals had particularly significant medial layer Cx37 immunostaining (Fig. 4F). Markedly reduced endothelial Cx37 in Cx40/ aorta was confirmed by en face immunostaining of thoracic aorta from 7-week-old wild-type and Cx40/ mice (Fig. 3Q-T). En face immunostaining of wild-type aorta resulted in fields where individual endothelial cells were partly or completely outlined by Cx37-containing punctae. By contrast, en face immunostaining of Cx40/ aorta resulted in a very weak, patchy Cx37 signal. Overexposure of photographic images (Fig. 3R, inset), confirmed residual levels of Cx37 present in Cx40/ endothelium. En face staining results for Cx37 were confirmed with the alternative Alpha Diagnostics Cx37 antibody (not shown). The presence of Cx40 and Cx43 in cryosections was also examined. Cx40 was detected exclusively in the endothelium of both wild-type and Cx37/ aortic sections (Fig. 3E,G,M,O). Although Cx40 immunostaining persisted in the Cx37/ endothelium, there was a significant decrease in the intensity of the signal, consistent with the reduction in Cx40 levels observed on western blots. Cx43 was not detected in the endothelial layer when aortic sections were immunostained with anti-Cx43 antibodies, but was detected in the media (not shown). Finally, we immunostained sections of abdominal aorta, iliac artery and coronary artery with anti-Cx37 and anti-Cx40 antibodies and obtained similar results (not shown). As in thoracic aorta, endothelial Cx37 signals were significantly reduced in all of the Cx40/ arteries examined. Thus, the dependence of endothelial Cx37 levels on the presence of Cx40 may be a general feature of the arterial system.
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Connexin immunostaining was also done with cryosections of E18.5 aortas (Fig. 3A-H). We hypothesized that Cx37 levels might be particularly low in embryonic Cx40/ aortas, given that western blots had suggested that the effect of eliminating Cx40 on Cx37 levels in aortic endothelium was age dependent, with younger postnatal animals showing greater declines in Cx37. Cx37 was in fact very difficult to detect in E18.5 Cx40/ aortic endothelium, although it was readily detected in E18.5 wild-type endothelium. By contrast, Cx40 immunostaining looked identical in Cx37/ and wild-type endothelium at this stage.
RT-PCR analysis
RT-PCR was performed to determine whether decreases in non-ablated connexin
levels were due to a decline in connexin mRNA levels
(Fig. 5). A lysate was
collected from thoracic aorta endothelium of 6-7-week-old mice by briefly
passing a lysis solution through the vessel lumen, and total RNA was purified.
After reverse transcription, specific primers were used to amplify segments of
Cx37, Cx40, Cx43 and GAPDH cDNAs. Amplicons were of the expected size and
depended on reverse transcription (Fig.
5A). Cx37 and Cx40 amplicons were absent from
Cx37/ and Cx40/ samples,
respectively, confirming the specificity of the PCR amplifications
(Fig. 5A). Semi-quantitative
RT-PCR was performed to compare the levels of non-ablated connexin transcripts
in wild-type, Cx37/ and
Cx40/ aortic endothelium
(Fig. 5B-D). Cx37 mRNA levels
were not significantly different in Cx40/ versus
wild-type aortic endothelium, nor was there a change in Cx40 mRNA in
Cx37/ versus wild-type aorta
(Fig. 5D). Amplification of
Cx43 signal from endothelial samples was weaker than for Cx37 and Cx40 and
more variable (Fig. 5B,D).
Differences in Cx43 mRNA levels (in Cx37/
endothelium, for example) did not reach statistical significance. Because Cx43
protein was not detected in aortic endothelium by western blotting or
immunostaining, Cx43 signals in the RT-PCR analysis may be the result of
unintended extraction of leukocytes during the RNA isolation procedure. We
determined that there was minimal contamination of our endothelial RNA
preparations with medial layer RNA by performing RT-PCR with primers for
smooth-muscle actin (SMA), comparing endothelial RNA fractions with whole
aorta RNA. Although SMA RNA was detected in the endothelial samples, the level
of SMA RNA in the endothelial samples was 500-fold lower than in an
equivalent length of whole aorta. Thus, contamination of endothelial RNA with
medial layer RNA was only
0.2%.
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Dye-transfer experiments
Dye injections were performed to examine the effects of connexin ablation
on interendothelial communication mediated by gap junctions. Segments of
thoracic aorta from wild-type, Cx37/,
Cx40/ and
Cx37+/Cx40/ mice were removed, and
endothelial cells were injected with a mixture of a gap-junction-permeable
tracer (Biocytin or NBD-TMA) and a fluorescently labeled dextran. The
fluorescent dextran, which is impermeable through gap junctions, was used to
mark the injected cell and as a control for transfer that was not mediated by
gap junctions. Biocytin transfer to neighboring cells was detected with
TMR-Neutravidin (Fig. 6),
whereas NBD-TMA could be detected directly because it is a fluorescent
compound (Fig. 7). After the
transfer period, the number of endothelial cells containing tracer was counted
and compared between genotypes. Biocytin transfer experiments were done with
aortas from mice aged 3 weeks, 6-7 weeks or 8-weeks to determine whether
there were age-related differences in the effect of connexin ablation on
intercellular communication. Transfer of biocytin occurred extensively in
wild-type endothelium (Fig.
6A). In aortas from 6-7-week-old mice, for example, the mean
number of labeled endothelial cells was 427±70 cells
(Fig. 10A)
(Table 2). The absence of Cx37
resulted in a small reduction (36%) in biocytin transfer at 3 weeks, a greater
reduction (78%) at 6-7 weeks and no significant reduction at
8 weeks
(Fig. 6C,
Fig. 10A)
(Table 2). Ablation of Cx40
resulted in a generally greater reduction in biocytin transfer compared with
Cx37 elimination, with the exception of aortas from 6-7-week-old mice, where
Cx40/ and Cx37/ aortas
exhibited about the same amount of transfer. Biocytin transfer was reduced by
73%, 72% and 62% in aortas from mice aged 3 weeks, 6-7 weeks and
8 weeks,
respectively, compared with wild-type mice
(Fig. 6E,
Fig. 10A)
(Table 2). Aortas from
Cx37+/Cx40/ mice showed a further
reduction in biocytin transfer, which was reduced by 85%, 88% and 83% in
aortas from Cx37+/Cx40/ mice aged 3
weeks, 6-7 weeks and
8-weeks mice, respectively, compared with wild-type.
NBD-TMA transfer was determined in aortas from animals that were
8 weeks
old (Fig. 7). No significant
change in NBD-TMA transfer was observed in Cx37/
versus wild-type aortas at this age (Fig.
7) (Table 2). The
absence of Cx40, however, reduced NBD-TMA transfer by 51%.
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Dye-transfer measurements were also performed with aortic endothelium that
lacked both Cx37 and Cx40.
Cx37/Cx40/ mice die
perinatally, so dye injections were done on aortas from E18.5 embryos
(Simon and McWhorter, 2002).
We confirmed that Cx37/Cx40/
embryos lacked both Cx37 and Cx40 in aortic endothelium
(Fig. 8A-D). Compensatory
changes in Cx43 expression were not observed in the double-knockout aortas
(Fig. 8E,F). Wild-type aortas
(C57BL6/129Sv strain) exhibited biocytin transfer to 275±62 cells at
this developmental stage (Fig.
9A, Fig. 10B)
(Table 2). Interendothelial
transfer of biocytin was eliminated, however, in aortas from
Cx37/Cx40/ E18.5 embryos of
the same strain background (Fig.
9D). Thus, only the injected cell was labeled with biocytin in
Cx37/Cx40/ aortas. The
absence of dye-transfer was not due to gross defects in aortic development, as
Cx37/Cx40/ aortas showed
normal expression of both endothelial and smooth-muscle-cell markers
(Fig. 8G-J). In addition,
silver nitrate staining of
Cx37/Cx40/ E18.5 aortas
revealed that endothelial cell morphology and cell-cell contacts were normal
(Fig. 8K,L). Finally, we
examined the effects of ablating only one of the endothelial connexins from
E18.5 aorta (Fig. 9,
Fig. 10B)
(Table 2). The absence of only
Cx37 did not have any effect on biocytin transfer at this embryonic stage
(Fig. 9B). Eliminating only
Cx40, however, did have a striking effect on the transfer of biocytin
(Fig. 9C). The number of
labeled cells was reduced by 96% and 98% in Cx40/ and
Cx37+/Cx40/ E18.5 aortas,
respectively. The effect of Cx40 ablation on dye-transfer was therefore much
more pronounced in the embryonic aorta than in the postnatal aorta.
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Long-term survival of
Cx37+/Cx40/ versus wild-type
mice
The long-term effects of a deficiency in endothelial communication have not
been previously investigated. To address this issue, the survivability of
Cx37+/Cx40/ versus wild-type mice
was compared over a two-year period (Fig.
11). We chose to examine
Cx37+/Cx40/ mice because aortas
from these animals exhibit lower levels of interendothelial dye-transfer than
either Cx37/ or Cx40/
aortas. Furthermore, dye-transfer is persistently reduced in
Cx37+/Cx40/ animals, even in older
ones. Cohorts of Cx37+/Cx40/ and
wild-type animals were housed under identical conditions for up to two years.
Although Cx37+/Cx40/ animals did
not show a sharp drop-off in viability, they began dying earlier than the
wild-type mice (Fig. 11).
Furthermore, after two years, only 13% of the
Cx37+/Cx40/ animals were alive,
compared with 42% of the wild-type animals. These data suggest that a
deficiency in endothelial communication may have long-term consequences on
vascular health and longevity.
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Discussion |
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The consequences of a deficiency in Cx37 versus Cx40 were substantially different with regard to interendothelial dye-transfer. Ablation of Cx40 generally had a greater effect on dye-transfer than ablation of Cx37, except in the 6-7 week-old age group, where the decline in transfer was about equivalent in Cx37/ and Cx40/ endothelium. In the other age groups, Cx40/ aortic endothelium exhibited significantly less dye-transfer than Cx37/ endothelium. Possibly, Cx40 is more abundant than Cx37 in the endothelium, but we did not attempt to compare the relative abundance of Cx37 and Cx40. Surprisingly, the effect of Cx40 deficiency on dye-transfer was age-dependent. In postnatal Cx40/ animals, dye-transfer was reduced to a greater extent in younger versus older animals. Continuing this trend, in Cx40/ aortas from E18.5 embryos, dye-transfer was drastically reduced in the absence of Cx40, with transfer occurring to only a few cells. By contrast, the elimination of Cx37 did not have any effect on biocytin dye-transfer in the embryonic endothelium. Thus, the effects on interendothelial communication of a deficiency in either Cx37 or Cx40 are not identical, and vary with developmental stage. Compensatory mechanisms may come into play in older postnatal animals to partially restore communication to more normal levels.
An important finding of this study is that targeted ablation of a specific
endothelial connexin not only eliminates the targeted connexin, it also
results in a substantial decrease in the levels of the non-ablated connexin.
Thus, Cx37 and Cx40 are mutually dependent on each other for optimal
expression in vascular endothelium. In particular, ablation of Cx40 resulted
in up to a 17-fold drop in Cx37 protein in aortic endothelium as determined by
western blotting. The drop in Cx37 was observed both with alkaline-extracted
membrane fractions, which enrich for gap-junction plaques, and with SDS sample
buffer lysates of endothelium. Thus, both total endothelial Cx37 and
plaque-associated Cx37 are affected by the absence of Cx40. In the reciprocal
case, ablation of Cx37 resulted in a decline in Cx40, but to a lesser extent;
elimination of Cx37 resulted in up to a 4.2-fold drop in Cx40 protein in
aortic endothelium. The dependency of Cx37 levels on the presence of Cx40 was
age-dependent, with greater reductions in Cx37 occurring in aortas from
younger versus older animals. This finding also suggests that age-dependent
compensatory mechanisms may be a factor in partially restoring communication
in older animals. Interestingly, age-dependent changes in endothelial connexin
expression and colocalization have been reported for rat aorta
(Yeh et al., 2000). Expression
of endothelial Cx37, for example, showed a downward trend with age, and the
extent of colocalization of Cx37 and Cx40 declined with age
(Yeh et al., 2000
). Therefore,
age-dependent declines in non-ablated connexins could be related, in part, to
changes in connexin expression in wild-type endothelium that occur with age.
Finally, we also observed a small decrease in endothelial caveolin-1 levels in
younger connexion-knockout mice. Recent evidence indicates that caveolin-1 can
interact with Cx43, as well as with Cx26, Cx32 and Cx46 in cultured cells
(Schubert et al., 2002
).
Possibly, caveolin-1 interacts with Cx37 and Cx40 in aortic endothelial cells
and its synthesis, targeting or stability could be affected by connexin
ablation.
Immunohistochemistry confirmed that decreases in non-ablated connexin levels occurred specifically in the endothelium of Cx37/ or Cx40/ aorta. Cx37 immunostaining was markedly reduced in the aortic endothelium of 7-week-old Cx40/ mice compared with wild-type controls. In addition, we observed a dramatic reduction in Cx37 immunostaining in aortas from E18.5 Cx40/ mice. This last result provides a molecular explanation for the sharp reduction in interendothelial dye-transfer that occurs in E18.5 endothelium in the absence of Cx40, as Cx37 levels were quite low in embryonic Cx40/ endothelium. In postnatal Cx40/ animals, especially older animals, endothelial Cx37 is reduced to a lesser extent than in E18.5 animals, relative to wild-type, and consequently, the effect on interendothelial communication is not as great.
Dependency of connexin levels on the presence of another connexin family
member is not unique to endothelial cells. Targeted ablation of connexin32 in
mice significantly reduced the levels of connexin26 in the liver, where they
are normally co-expressed in hepatocytes, and also reduced total gap
junctional plaque area in the liver by 1000-fold
(Nelles et al., 1996). In
another recent example, deletion of connexin46 (Cx46) eliminated the
expression of connexin50 (Cx50) from the lens nucleus, where Cx46 and Cx50 are
normally co-expressed by lens fibers (Rong
et al., 2002
). Interestingly, ablation of Cx50 did not eliminate
Cx46 in the lens nucleus. This result is similar to what we observed with Cx37
and Cx40 in endothelial cells, with Cx37 being much more dependent on the
presence of Cx40 than vice versa. Finally, in Cx43-deficient hearts, Cx45
immunostaining at cardiac gap junctions was markedly reduced, although total
levels of Cx45 were unchanged (Johnson et
al., 2002
). Thus, in responses to connexin deletions, decreases in
co-expressed, non-ablated connexins seem to be a more common occurrence than
compensatory upregulation. These findings may be of some importance with
regard to understanding the pathology of connexin mutations in humans,
especially in tissues that express multiple connexins.
Changes in the levels of non-ablated connexins in the endothelium could be
due either to alterations in the efficiency of translation of connexin mRNA or
connexin processing, or to changes in connexin stability. Our RT-PCR
experiments indicated no significant changes in Cx37 mRNA levels in
Cx40/ aortic endothelium, nor were there changes in
Cx40 mRNA in Cx37/ endothelium. Therefore, it is
unlikely that the reduction in non-ablated connexins is due to decreased
transcription of the connexin genes. Possibly, Cx37 and Cx40 contribute to
heteromeric channels of mixed connexin content, which might be more stable
than homomeric channels. According to this model, eliminating one connexin
would permit the assembly of only less-stable homomeric channels, and
therefore levels of the non-ablated connexin would fall. Heteromeric channels
containing two connexins have been shown for several connexins by both
biochemical and electrophysiological methods
(Stauffer, 1995;
Jiang and Goodenough, 1996
;
Brink et al., 1997
;
He et al., 1999
). In addition,
Cx37 and Cx40 have been shown to colocalize to the same gap junction plaques
in endothelial cells (Yeh et al.,
1998
; Ko et al.,
1999
). Finally, the presence of both Cx37 and Cx40 could be
important for normal clustering of gap-junction channels into plaques in the
endothelial plasma membrane. Inefficient clustering in the absence of Cx40,
for example, might reduce the stability of Cx37-containing channels. Recently,
it was suggested that Cx40 could contribute to plaque structure by interacting
with proteins linked to the cytoskeleton
(Krüger et al.,
2002
).
Our results are substantially different from those published recently by
Krüger et al., who investigated an independently generated
Cx40/ mouse line
(Krüger et al., 2002).
Those investigators concluded that there was upregulation of Cx37 in the
aortic endothelium of Cx40/ mice rather than the
downregulation of endothelial Cx37 we describe here. They presented western
blot data that documented a
fourfold increase in Cx37 protein in
Cx40/ thoracic aorta, as well as a
twofold
increase in Cx43. The western blot data obtained by Krüger et al.,
however, might not accurately represent Cx37 levels in the endothelium, as
they collected protein from the entire aortic segment, including the medial
layer. In this study, we analyzed separately connexins from whole aorta,
endothelium-only fractions or medial layer-only preparations on western blots.
This methodological difference is relevant because we detected some Cx37 in
the medial layer by western blotting and by immunostaining. Cx37 has
previously been reported in the media of rat aorta, and most recently in the
caudal artery of the rat, where it was found to be more abundant than Cx43 or
Cx45 (Nakamura et al., 1999
;
Rummery et al., 2002
). The
amount of Cx37 reported in the medial layer varies with different arteries,
however, and appears to be lower in rat thoracic aorta than caudal artery
(Rummery et al., 2002
). Final
confirmation of the presence of Cx37 at medial smooth-muscle gap junctions
awaits careful analysis by immunogold electron microscopy. In this study, we
found that medial layer Cx37 signal was significantly elevated in
Cx40/ aortas as detected by both western blotting and
immunostaining. In this regard, Cx37 in the medial layer behaves similarly to
Cx43, which we find is also elevated in Cx40/ media,
in agreement with Krüger et al.
(Krüger et al., 2002
).
Possibly, medial Cx37 and Cx43 both increase in response to hypertensive
changes described in Cx40/ animals
(de Wit et al., 2000
). In some
experimental models of hypertension in the rat, expression of aortic Cx43 was
shown to increase by
twofold, similar to the
fourfold increase in
medial Cx43 and medial Cx37 we observed in Cx40/
aorta (Haefliger et al.,
1997
). The specificity of the medial layer Cx37 immunosignal was
confirmed by immunostaining Cx37/ aorta, which showed
no medial layer staining. Therefore, the western blot results of Krüger
et al. were possibly affected by changes in the levels of Cx37 present in the
medial layer as well as in the endothelial layer. Indeed, when whole aorta
preparations were analyzed by western blotting, we did not see the decline in
Cx37 in Cx40/ aortas that was observed with
endothelium-only preparations.
Our immunostaining results also differ from those presented by Krüger
et al., who reported that Cx37 immunosignals were distributed more
homogeneously in Cx40-deficient versus wild-type endothelium, so that areas
without Cx37 staining were less frequently observed in the
Cx40/ aorta
(Krüger et al., 2002). By
contrast, we observed a very substantial decrease in Cx37 immunostaining in
Cx40/ endothelium, both in cross sections and by en
face staining. These results are fully consistent with our western blot data.
The reasons for the different immunostaining data obtained in the study by
Krüger et al. and in this study are not clear. Differences in mouse
strains and ages or antibodies used in the studies might account for some
differences. In addition to the western blot and immunofluorescence data, our
RT-PCR data do not support the idea that there is an increase in endothelial
Cx37 gene expression when Cx40 is ablated. Finally, Krüger et al. found
very few gap junction plaques in the intima of Cx40/
mice when aortic sections were analyzed by electron microscopy
(Krüger et al., 2002
).
They suggested that aggregates of Cx37 might be too small to be seen in the
electron micrographs, but could be detected by immunofluorescence.
Alternatively, the scarcity of morphologically identifiable gap junctions in
the Cx40/ endothelium could be taken to support our
findings of a substantial reduction in endothelial Cx37 levels in the absence
of Cx40.
One might expect Cx40/ endothelium to exhibit
diminished coupling, especially since ablation of Cx40 also reduces
endothelial Cx37. In the present study, we found that biocytin transfer was
substantially lower in Cx40/ endothelium compared
with wild-type endothelium. Krüger et al. also compared dye-transfer
levels in Cx40/ versus wild-type aortic endothelium
(Krüger et al., 2002).
They reported extensive transfer of neurobiotin but not of Lucifer Yellow in
Cx40/ endothelium, whereas transfer of both tracers
occurred efficiently in wild-type endothelium. On the basis of these results,
they suggested that Cx37 channels have more restricted gating properties than
Cx40 channels. Previous studies have indicated that negatively charged tracers
diffuse poorly through Cx37 channels in some cell types, perhaps explaining
weak Lucifer Yellow transfer in Cx40/ aorta, since
Lucifer Yellow has a charge of 2
(Veenstra et al., 1994
).
Potentially, differences in the physical properties of neurobiotin versus
biocytin could account for the different dye-transfer results obtained in this
study, given that biocytin is structurally similar, but not identical to
neurobiotin. Neurobiotin has a molecular mass of 287 Da and carries a positive
charge at physiological pH, whereas biocytin is 372 Da and is electrically
neutral. However, we also observed a decrease in transfer of NBD-TMA, which
has a molecular mass of 266 Da and carries a positive charge: properties that
are similar to neurobiotin. Thus, it is unclear why
Cx40/ endothelium would exhibit decreased transfer of
biocytin and NBD-TMA, but not of neurobiotin.
What are the consequences of deficient endothelial communication on
vascular health? Mice that completely lack both Cx37 and Cx40 are not viable
beyond the first postnatal day and exhibit severe vascular abnormalities
(Simon and McWhorter, 2002).
Cx37/Cx40/ mice have
localized hemorrhages in skin, testis, gastrointestinal tissues and lungs, as
well as blood vessel dilatation and congestion. Moreover, vascular
dysmorphogenesis is evident in testis and intestine of
Cx37/Cx40/ animals. Thus,
endothelial communication is required for the normal development and/or
functional maintenance of portions of the mouse vasculature. The survival data
presented here suggest that even with connexin-deficient animals that retain
low levels of endothelial coupling, there may be a reduction in long-term
survival. After two years, there was a substantial difference in the
percentage of surviving Cx37+/Cx40/
animals versus wild-type animals.
Cx37+/Cx40/ animals, which have
80% reduction in aortic endothelial dye-transfer, may therefore serve as
a good animal model for studying the long-term as well as short-term
physiological effects of compromised endothelial communication.
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