Smooth Muscle–Targeted Knockout of Connexin43
Enhances Neointimal Formation in Response to
Vascular Injury
Yongbo Liao, Christopher P. Regan, Ichiro Manabe, Gary K. Owens, Kathy H. Day,
Dave N. Damon, Brian R. Duling
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Objective—Vascular disease alters and reduces connexin expression and a reduction in connexin 43 (Cx43) expression
diminishes the extent of atherosclerosis observed in a high-cholesterol diet murine model. We hypothesized that
connexins might play a role in the smooth muscle cell response to vascular injury.
Methods and Results—We therefore studied a line of smooth muscle cell-specific, Cx43 gene knockout mice (SM Cx43
KO) in which the carotid arteries were injured, either by vascular occlusion or by a wire injury. In the SM Cx43 KO
mice both types of injury manifested accelerated growth of the neointima and of the adventitia. Isolated vascular smooth
muscle cells from the SM Cx43 KO mice grew at a slightly faster rate in culture, and to marginally higher saturation
densities than those of control mice, but these changes were not adequate to explain the large changes in the injured
vessels.
Conclusions—These observations provide direct evidence that smooth muscle Cx43 gap junctions play a multi-faceted role
in modulating the in vivo growth response of vascular smooth muscle cells to vascular injury. (Arterioscler Thromb
Vasc Biol. 2007;27:1037-1042.)
Key Words: adventitia 䡲 atherosclerosis 䡲 smooth muscle 䡲 thrombus
I
ntercellular communication mediated by gap junctions
plays a pivotal role in the cardiovascular system, being
involved in such diverse processes as determination of
vasomotor tone, cell differentiation, growth control, embryonic development, and coordination of contraction of cardiac
muscle cells.1– 4 Gap junctions are formed from combinations
of 1 or more of the 4 connexin protein isomers that are known
to be expressed in the vasculature: Cx37, 40, 43, and 45,5–10
and there is accumulating evidence indicating that the connexins may play a role in a variety of vascular pathologies
including: hypertension,11–14 ischemia/reperfusion injury,15
and atherosclerosis.16 –22 Moreover, gap junctional communication is reduced in proliferating vascular smooth muscle
(VSM) cells,23 suggesting that the connexins might play a
role in the modulation of the vascular response to injury or
experimental atherogenesis.24
Our studies tested the hypothesis that selective deletion of
a particular connexin in VSM would modify the response to
vascular injury. To accomplish this, we generated a knockout
mouse in which deletion of the Cx43 gene was confined to
smooth muscle (SM) cells.25 Here we report the effects of this
deletion on the response of the carotid artery to injury, and on
the modification of the growth pattern of cultured VSM cells
isolated from the SM Cx43 knockout (KO) mice.
Materials and Methods
Generation of Mice With SM Cx43 KO
A line of mice in which the second exon of the Cx43 gene was
flanked by loxP sites was produced26 and crossed with a second line
of transgenic mice that carried a transgene composed of the SM
myosin heavy chain promoter/enhancer and the Cre recombinase
gene to induce smooth muscle-restricted Cre expression.25 Crossing
these 2 lines generated mice in which the Cx43 gene was deleted
from those cells in which myosin heavy chain drove the Cre
expression, ie, in SM cells, and resulted in cell specific deletion
(supplemental Figure II, available at http://atvb.ahajournals.org).
Mice homozygous for the floxed Cx43 gene were used as controls
for these experiments (supplemental Figure I). Breeding, housing,
maintenance, and experimental procedures were all conducted in
accordance with the approved practices of the University of Virginia
Animal Care and Use Committee.
DNA Preparation and Analysis
The genotype of each animal was confirmed by polymerase chain
reaction analysis using primer sequences described online (see
http://atvb.ahajournals.org).
Original received July 7, 2006; final version accepted February 14, 2007.
From the Department of Anesthesiology (Y.L.), Department of Molecular Physiology and Biological Physics (G.K.O., D.N.D., B.R.D.), and
Cardiovascular Research Center (K.H.D.), University of Virginia, Charlottesville, Va; Department of Pharmacology (C.P.R.), Merck Research
Laboratories, West Point, Pa; Department of Cardiology (I.M.), University of Tokyo, Tokyo, Japan.
Correspondence to Dr Brian R. Duling, Department of Molecular Physiology and Biological Physics, University of Virginia, School of Medicine, MR-4
Building, Room 6051, Charlottesville, VA 22908. E-mail brd@virginia.edu
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1037
DOI: 10.1161/ATVBAHA.106.137182
1038
Arterioscler Thromb Vasc Biol.
May 2007
Measurement of Blood Pressure and Heart Rate
Tail cuff measurements of systolic blood pressure and heart rate were
obtained using a Visitech Systems tail cuff instrument.26 As shown
in supplemental Table I, there was no significant difference in blood
pressure of the controls compared with the SM CX43 KO animals.
Occlusion Injury of the Carotid Artery
A common carotid artery injury was produced by ligation as
described by Kumar et al.27 Although the vessel responses to
occlusion were altered in the carotids of the SM Cx43 KO mice
(compare supplemental Figures IIIB and IIIF with IIID and IIIH), the
changes were less predictable than those seen in the wire injury
model; therefore, we selected the latter treatment for the detailed
quantitative analysis.
Denudation of the Carotid Artery Using a
Guide Wire
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A second type of carotid artery injury was made as described by
Lindner.28 After anesthesia, a transverse arteriotomy was made in the
left external carotid artery, and a 0.014-inch flexible angioplasty
guide wire (Advanced Cardiovascular Systems, Inc, Temecula,
Calif) was introduced and advanced ⬇1 cm toward the aortic arch.
The intima of the left common carotid artery was injured by rotating
the wire 3 times during withdrawal and the left external carotid artery
was then tied off. The right external carotid artery was ligated as a
control. Mice were allowed to recover and returned to the animal
care facility.
Tissue Harvest and Morphological Examination
Carotids were obtained from anesthetized mice, fixed, and morphometrics performed to determine: lumen radius, thickness of media,
neointimal area, luminal area, medial area, and adventitial area
(supplemental Table I).
Immunohistochemistry
Paraffin sections were examined on an Olympus Fluoview, duallaser, confocal microscope. Specificity of antibodies was confirmed
by preincubation of each antibody with the appropriate peptide.
Rabbit anti-mouse Cx43 antibody was purchased from Alpha Diagnostic International (Cx43B12-A; San Antonio, Tex). Rabbit antihuman von Willebrand factor antibody and mouse monoclonal SM
anti-␣ actin antibody were obtained from Sigma (St. Louis, Mo). Rat
monoclonal anti-CD45 antibody was purchased from BD Biosciences (San Diego, Calif). Further details of immunohistochemistry are described in the online Methods section.
Growth Curve of Cultured VSM Cells
VSM cells were isolated from of 3 to 4 aortas (4 to 5 weeks old, male
or female) pooled and placed into culture following the protocol of
Rovner et al.29 Only passages 3 to 5 were used for the study. The
VSM cells were characterized by immunostaining for SM myosin
heavy chain25 and SM-␣ actin (Sigma). At daily intervals, plates
were trypsinized to release the cells, which were then counted in
triplicate on a hemocytometer.
Statistics
All values are expressed as mean⫾SEM. One-way ANOVA was
used for statistical analysis of group comparisons. P⬍0.05 was
considered significant.
Results
Production and Characterization of the
SM-Specific Cx43 KO Mice
Mice homozygous for both the floxed Cx43 allele and the
myosin heavy chain-Cre gene grew normally and were fertile.
Evidence demonstrating the specificity and efficacy of the
deletion produced in these mice is presented (supplemental
Figures I and II). Cx43 immunostaining was punctuate in the
media of arteries from control animals and was greatly
reduced in the VSM of SM Cx43 KO mice (Figures 1 and 2,
and supplemental Figures IIA to IIE and IIIE, IIIF). These
observations are consistent with the demonstration by Regan
et al25 that Cre expression in the myosin heavy chain–Cre
transgenic animals is uniform, and is restricted to the SM
cells and thus guides cell-specific deletion.
Unexpectedly, the intensity of Cx43 immunostaining in the
endothelium of the aorta from the SM Cx43 KO mice was
also often reduced or even eliminated in the SM Cx43 KO
animals (compare supplemental Figure IIB and IIE). Endo-
Figure 1. Comparison of injury responses of control (A to G) and SM Cx43 KO (B to H) mice to wire injury of the left carotid artery. A
and B, Sham-injured, right carotid arteries. C to H, Wire-injured left carotid arteries. A to F, Hematoxylin and eosin stain to visualize the
neointima formation and the adventitial proliferation at different magnifications. G and H, Confocal fluorescent images used for the
measurement of internal elastic lamina and external elastic lamina. White arrowheads show the locations of the internal elastic lamina.
A to D, bar⫽200; E to H, bar⫽100.
Liao et al
Connexins and Arterial Injury
1039
Figure 2. Identification of cell types in
the wire-injured carotid arteries. All sections are from the wire-injured carotid
arteries of SM Cx43 KO mice at 14 days
after surgery. Cells in the neointima and
media were negative for Cx43 (A). B to
D, ␣-Actin, von Willebrand factor, and
CD45 demonstrate that most of the cells
in the neointima are SM cells, although
endothelial cells are present (*, C) and
leukocytes are diffusely distributed in the
adventitia (#, D). White arrows show the
locations of IEL (bar⫽50 um.).
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thelium was still present in the vessels of SM KO mice as
evidenced by the presence of platelet endothelial cell adhesion molecule (PECAM) (supplemental Figure IIB, IIE) and
von Willebrand factor labeling (Figure 2B).
Blood Pressure and Heart Rate
The SM Cx43 KO mice showed no alteration in blood
pressure or heart rate (supplemental Table I).
Enhanced Proliferation of VSM After Injury in
the SM Cx43 KO Mice
Examples of observations made on the carotids of control and
SM Cx43 KO mice before and after wire injury are shown in
Figure 1. After sham operation, there was no significant
difference between the control and SM Cx43 KO mice
(compare Figure 1A and 1B). At 7 days after surgery, no
neointima formation was observed in the wire-injured carotid
artery of control mice, although there was modest growth in
adventitia (compare Figure 1A and 1C). SM Cx43 KO, mice
showed substantial increase in both neointima and adventitia
(compare Figure 1B and 1D). Quantitative measurements
confirmed the impression given in Figure 1, with morphological measurements showing significant increases in areas of
the neointima, media, and adventitia when the wire-injured
carotid arteries of control and SM Cx43 KO mice were
compared (Table). Immunostains to determine cell types
associated with the changes in vessel wall morphology
shown in Figure 1 are presented in Figure 2. Cells in the
neointima are, for the most part, SM-like (Figure 2C),
although there are occasional endothelial cells (Figure 2B).
Leukocytes are occasionally present in the adventitia but
not evident in the neointima. Occasional Cx43-positive
cells can be seen in the adventitia (Figure 2A).
rates. Cells isolated from mouse aorta were confirmed to be
VSM by positive staining for SM myosin heavy chain and
SM ␣-actin (supplemental Figure IVA, IVB). Cultured VSM
cells from the SM Cx43 KO mice exhibited a VSM phenotype similar to the control cultured cells, and grew logarithmically, although at a slightly faster rate and to a somewhat
higher confluent density than those harvested from the
control mice (online data, supplemental Figure IVC). Although the deletion of Cx43 gene suggested a slightly
elevated proliferation of the VSM, the change in vitro was not
nearly as great as that observed in vivo.
Discussion
Our findings support and extend the previous evidence that
connexin expression plays an important role in regulation of
vascular growth in response to injury.16,18,20,24,30 –33 Neointimal formation after injury was markedly increased in SM
Cx43 KO mice as compared with controls, and VSM cells
derived from the KO mice showed slightly enhanced proliferation and density in vitro. These results are consistent with
previous studies implicating a role for gap junctional comMorphological Analysis of Carotid Arteries After Wire Injury
Genotype
Neointimal area (⫻103)
We compared the growth characteristics of control and Cx43
KO VSM cells in culture to evaluate their intrinsic growth
0
SM Cx43 KO Mice
(n⫽9)
8.5⫾3.2*
Lumen area (⫻103)
96.2⫾5.3
Medial area (⫻103)
19.3⫾2.1
29.0⫾3.5*
Adventitial area (⫻103)
17.9⫾7.0
109.8⫾19.5†
Lumen radius (⫻103)
Thickness of media (⫻103)
Growth Rates of VSM From Control and SM
Cx43 KO Mice
Floxed Cx43 Mice
(n⫽8)
114.2⫾11.7
174.6⫾4.7
188.8⫾9.5
16.8⫾1.8
22.3⫾2.7
Perimeters of lumen, internal elastic lamina, and external elastic lamina
were measured from confocal images. Radius and thickness are measured in
␮m. Area is measured in ␮m2. Data are expressed as mean⫾SE.
*P⬍0.05; †P⬍0.01.
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Arterioscler Thromb Vasc Biol.
May 2007
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munication in regulation of cell growth, presumably through
cell– cell-mediated transfer of signaling molecules or electrical signaling. Our findings suggest that the absence of intact
Cx43 signaling disrupts critical feedback control pathways
necessary for vascular morphogenesis, as has been shown for
the endothelium.34
The success of using the Cre/loxP system in experiments
such as these is dependent on 2 factors: (1) that the insertion
of loxP does not interfere with the native gene expression;
and (2) that the promoter driving Cre expression is cellspecific. Polymerase chain reaction analysis revealed no
deletion of the Cx43 allele in the brain gray matter or white
blood cells, samples that should contain no SM cells (supplemental Figure I). Most importantly, we examined the efficiency and selectivity of Cx43 deletion in VSM layers by
immunohistochemistry. As shown in Figure 2 and supplemental Figure II, the Cx43 immunostain in VSM of aorta and
carotid artery was markedly reduced in the media of the SM
Cx43 KO mice compared with controls.
Cx43 does not appear to be completely eliminated in the
media of the KO mice, presumably reflecting incomplete
gene deletion with the cre system. It is noteworthy that
cre-based deletion need not be complete if some of the cells
fail to express cre at a critical time in development. This is a
fact that has received very little experimental scrutiny by
those who use the conditional deletion approach and we hope
to analyze cre expression in more detail in the future.
Cx43 Gap Junctions Are Critical for the
Remodeling Process in Response to
Vascular Injury
Restenosis is a combination of neointimal formation and
arterial remodeling involving alterations in many processes,
including complex interactions among endothelium, SM
cells, fibroblasts, and inflammatory cells. Thus, our observations of a striking difference in the intimal growth response
between the wild-type and the SM Cx43 KO animals in
response to wire injury (Figure 1, Table) are both novel and
of great potential significance. The data suggest that disruption of normal gap junctional communication might contribute to other disease states associated with abnormal VSM
growth, including hypertension, atherosclerosis, and postangioplasty restenosis, and that the gap junctions might serve as
targets for new therapeutic interventions for these human
diseases.27,35–37
Our findings are in sharp contrast to the work of Chadjichristos
et al,38 who showed that heterozygous Cx43 KO mice manifested reduced neointimal formation rather than enhanced
neointimal formation. Their experimental model included a
high-fat diet, thus differences may simply reflect different
vascular adaptive processes. In addition, the mice used in the
studies by Chadjichristos were global KO mice, thus Cx43
was reduced in all cell types expressing Cx43, and as a result
we do not know which cell type initiated and which contributed to the altered atherosclerotic response. The differences
between the results in the 2 experiments may reflect complex
interactions between different cell types in the global KO.
An incidental but striking finding was that the adventitial
area of injured carotid arteries in the SM Cx43 KO mice was
6-times greater than that of control mice (Table, Figure 1F),
despite the evidence that the activity of the SMC promoter is
restricted to the VSM25 (supplemental Figure I), suggesting
that the effects of Cx43 deletion on adventitial growth were
secondary to some change in the SM. Deletion of Cx43 from
the VSM might alter the release of paracrine growth factors
such as platelet-derived growth factor (PDGF) BB or basic
fibroblast growth factor (bFGF), which could be mitogenic
for adventitial fibroblasts as well as VSM. Studies by others
have shown adventitial reactions in atherosclerosis, arteritis,
and after angioplasty.39,40 Moreover, Booth et al41 reported
that manipulation of the vessel wall resulted in lesions that
mimic the biochemical and morphological changes observed
in early stages of human atherosclerosis. It is thus possible
that disruption of Cx43 signaling in VSM in some manner
exacerbates vascular inflammation in response to injury and
thereby secondarily alters adventitial cell growth.
There are controversial reports suggesting that after injury
there is migration of adventitial fibroblasts toward the media
and that these cells subsequently contribute to the neointima
formation.42,43 In addition, it has been shown that there are
pluripotent cells in the adventitia, which may contribute to the
growth of the media.44 Perhaps the myofibroblasts within the
adventitia were actually derived from the migration of medial
SM cells into the adventitia, where they may have undergone
phenotypic switching to a fibroblast-like cell.45 However, it
has generally been assumed that there is migration into the
intima, not into the adventitia. The positive immunostaining
with ␣-actin and CD45, and negative staining with Cx43
antibodies (Figure 2) are consistent with the possibility of
migration of medial cells into the adventitia.
Identification of Cell Types in the Neointima
We used multiple antibodies to identify the cell types in the
neointima and found that the majority of neointimal cells
stained positive for ␣-actin, although some cells at the
luminal surface stained positive with the endothelial cellspecific marker von Willebrand factor. Few CD45-positive
cells were seen in the neointima, although CD45-positive
cells were diffusely distributed in the adventitia. The majority
of the intimal cells express ␣-SM actin and were thus likely
VSM cells (Figure 2B). Negative Cx43 immunostaining of
SM cells in the neointima area is consistent with the idea that
the augmented neointimal formation in SM Cx43 KO mice
was primarily caused by migration of medial SM cells that
had undergone Cx43 gene deletion through the Cre/loxP
interaction, and not the consequence of secondary alterations
in vascular inflammation. These findings are reminiscent of
the findings of Kwak et al21 in atherosclerotic mice.
Interplay of Cx43 Expression Between VSM
and Endothelium
Although our data indicate that the Cre-mediated deletion
was confined to a single gene in a single cell type, secondary
interactions appeared to cause a reduction in the Cx43 protein
in the aortic endothelium (compare supplemental Figure IIB
and IIE). The PECAM and von Willebrand factor antibody
staining indicated that the endothelial cells were still present
in the KO mice (Figure 2 and supplemental Figure II). In a
Liao et al
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previous report we noted that mice with a cell-specific
deletion of the endothelial cell Cx43 showed a reduction of
Cx43 message in the adjacent VSM layers.26 Such a process
previously reported in the liver by Nelles et al,46 and the
parallel changes in the Cx43 expression in endothelium of the
SM CX43 KO mice emphasize that caution should be used in
the interpretation of the specificity of conditional KO experiments, especially when dealing with a protein that is part of
a complex.
Coregulation of the Cx43 proteins in the 2 cell types might
reflect paracrine signaling or, alternatively, such coregulation
might arise as a result of transcellular signaling mediated by
myoendothelial junctions. Simon47 observed such coregulation of Cx40 and 43 in Cx40 KO mice, and thus his findings
support a linkage in the mixture of gap junctional proteins
expressed, at least in endothelium. As yet, there has been no
demonstration of a functional myoendothelial junction in the
mouse aorta, although one has been shown in a coculture
system.48
In summary, we provide clear evidence that Cx43 signaling
in VSM plays a key role in regulation of neointimal growth
after vascular injury. The enhancement of in vitro growth
properties of cultured aortic VSM derived from SM Cx43 KO
mice provides additional evidence that Cx43 plays a role in
growth regulation of VSM, but the much smaller in vitro
effect argues that additional factors are involved in the
determination of the sensitivity of the VSM to injury in vivo.
Moreover, the alterations in both intima and adventitia
suggest that overall vascular remodeling and morphogenesis
are dependent on Cx43 signaling through as yet poorly
understood processes.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Sources of Funding
21.
This work was supported by NIH grants HL12792, HL23531, and
HL53318 (B.R.D.); grants R01 HL57353 and P01 Hl19242
(G.K.O.), the Academic Enhancement program on Gene Transfer
and Gene Therapy, and the Robert M. Berne Cardiovascular Research Center at the University of Virginia.
22.
Disclosure
None.
23.
24.
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Downloaded from http://atvb.ahajournals.org/ by guest on September 30, 2016
Smooth Muscle−Targeted Knockout of Connexin43 Enhances Neointimal Formation in
Response to Vascular Injury
Yongbo Liao, Christopher P. Regan, Ichiro Manabe, Gary K. Owens, Kathy H. Day, Dave N.
Damon and Brian R. Duling
Arterioscler Thromb Vasc Biol. 2007;27:1037-1042; originally published online March 1, 2007;
doi: 10.1161/ATVBAHA.106.137182
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