Progressive vascular smooth muscle cell defects
in a mouse model of Hutchinson–Gilford
Renee Varga*, Maria Eriksson†, Michael R. Erdos*, Michelle Olive*, Ingrid Harten‡§, Frank Kolodgie¶, Brian C. Capell*,
Jun Cheng?, Dina Faddah*, Stacie Perkins*, Hedwig Avallone¶, Hong San*, Xuan Qu*, Santhi Ganesh*,
Leslie B. Gordon*,**, Renu Virmani¶, Thomas N. Wight‡§, Elizabeth G. Nabel*††, and Francis S. Collins*‡‡
*Genome Technology Branch and?Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, 50 South
Drive, Bethesda, MD 20892;†Department of Medical Nutrition, Karolinska Institutet, Novum, Halsovagen 7, Hiss E, Plan 6, 141 57 Huddinge, Sweden;
‡Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, WA 98101-2795;§Department of Pathology, University of Washington
School of Medicine, Seattle, WA 98195;¶CVPath, Inc., 19 Firstfield Road, Gaithersburg, MD 20878; **Department of Pediatrics, Brown Medical School,
Providence, RI 02912; and††National Heart, Lung, and Blood Institute, National Institutes of Health, 31 Center Drive, Bethesda, MD 20892
Contributed by Francis S. Collins, January 2, 2006
Children with Hutchinson–Gilford progeria syndrome (HGPS) suf-
fer from dramatic acceleration of some symptoms associated with
normal aging, most notably cardiovascular disease that eventually
leads to death from myocardial infarction and?or stroke usually in
point mutation in the lamin A (LMNA) gene is the cause of HGPS.
This missense mutation creates a cryptic splice donor site that
produces a mutant lamin A protein, termed ‘‘progerin,’’ which
carries a 50-aa deletion near its C terminus. We have created a
mouse model for progeria by generating transgenics carrying a
human bacterial artificial chromosome that harbors the common
HGPS mutation. These mice develop progressive loss of vascular
smooth muscle cells in the medial layer of large arteries, in a
pattern very similar to that seen in children with HGPS. This mouse
this devastating disorder and for exploring cardiovascular disease
lamin A ? atherosclerosis ? laminopathy
disease postnatally (see Progeria Research Foundation’s medical
and research database at www.progeriaresearch.org). Progressive
facial disproportion, mandibular, and clavicular hypoplasia, and
osteoporosis (1). The most serious aspect of the disease, however,
and the cause of death in ?90% of cases, is rapid, progressive
arterial occlusive disease, with death from myocardial infarction or
stroke occurring at an average age of 13 years (range, 8–21 years).
Although autopsy data on progeria patients is very limited,
consistent pathologic findings have been reported in the arterial
system (2–5). Specifically, postmortem studies have identified pro-
found loss of vascular smooth muscle cells (VSMC) in the medial
layer of large arteries, such as the aorta and carotid arteries, with
these medial changes have been generalized features of atheroscle-
rosis with focal areas of calcification. The interlaminar spaces have
been reported to contain thin and disorganized collagen fibrils as
well as cell debris or matrix vesicles. Cholesterol clefts are lacking,
and children with progeria have normal lipid profiles.
HGPS is caused in nearly all classic cases by a de novo mutation,
codon 608 remains glycine), in exon 11 of the lamin A?C gene on
syndrome have also been traced to other mutations in the LMNA
gene (6, 8–12). There are at least eight other diseases in addition
to HGPS caused by LMNA mutations, collectively known as the
hildren with Hutchinson–Gilford progeria syndrome (HGPS)
appear normal at birth, but begin to display features of the
lipodystrophy, mandibuloacral dysplasia, Charcot–Marie–Tooth
disorder-type 2, dilated cardiomyopathy-type 1A, and Emery–
have been found in connection with these various laminopathies.
The three normal protein products of the LMNA gene, lamin A,
(14). The nuclear lamina is located just under the inner nuclear
membrane and plays significant roles in nuclear shape, DNA
replication and transcription, cell division, and chromatin organi-
zation. Lamin A is posttranscriptionally modified. The initial pre-
cursor, prelamin A, undergoes a series of processing steps that
include farnesylation of the cysteine in the C-terminal CAAX
sequence, cleavage of the terminal AAX sequence with addition of
a methoxy group to the terminal cysteine, and subsequent cleavage
of the terminal 15 aa by the protease ZMPSTE24. The HGPS
mutation in exon 11 creates a novel splice donor site 150-bp
upstream of the true exon 11 splice donor site. The protein that is
50 aa near the C terminus. Within these 50 aa is the ZMPSTE24
recognition site. Progerin is thus unable to be cleaved, resulting in
a permanently farnesylated form of lamin A (6, 15). We hypothe-
sized that the retention of the farnesyl group forces progerin to
remain embedded in the nuclear membrane and form multimeric
complexes with mature wild-type lamin A and other proteins,
creating a mislocalized multiprotein complex that alters nuclear
structure and function. In support of this hypothesis, mutations in
is phenotypically similar to HGPS (16).
Several useful mutant Lmna mouse models have been created
produces a cardiac and skeletal myopathic phenotype similar to
human Emery–Dreifuss muscular dystrophy (17). Mounkes et al.
(18) also reported a knock-in of the L530P mutation, associated in
humans with autosomal dominant Emery–Dreifuss muscular dys-
wild-type (WT) mice, but homozygous mice have a phenotype
resembling HGPS, with severe growth retardation within 4–5 days
and death within 4–5 weeks. Features that these mice share with
children with HGPS include micrognathia and abnormal dentition,
Conflict of interest statement: No conflicts declared.
Abbreviations: HGPS, Hutchinson–Gilford progeria syndrome; VSMC, vascular smooth
muscle cells; WT, wild type; BAC, bacterial artificial chromosome; PG, proteoglycan; NP,
‡‡To whom correspondence should be addressed at: National Human Genome Research
Institute, National Institutes of Health, Building 31, Room 4B09, 31 Center Drive,
MSC2152, Bethesda, MD 20892-2152. E-mail: email@example.com.
© 2006 by The National Academy of Sciences of the USA
February 28, 2006 ?
vol. 103 ?
absence of the s.c. fat layer, sclerodermal-like increases in collagen,
decreased bone density, malformation of the scapulae, and hypo-
gonadism. Nevertheless, these mice have no obvious defects in the
the knock-in allele actually causes a more complex splicing anom-
aly. Yang et al. (19) have recently reported a knock-in model whose
mutant Lmna allele codes exclusively for progerin, without any
bone disease, but detailed phenotypic characterization of the
vascular system has not yet been reported for these animals.
We created a transgenic mouse that carries the G608G mutated
human LMNA on a 164-kb bacterial artificial chromosome (BAC).
Although this mouse lacks the external phenotype seen in human
progeria, it does demonstrate the progressive vascular abnormali-
ties that closely resemble the most lethal aspect of the human
Using a two-step recombination protocol in Escherichia coli (20),
HGPS G608G mutation (Fig. 1). G608G transgenic mice were
created by injecting BAC DNA, both as a circular construct and
after linearizing with a unique PI-SceI restriction site. As a control,
transgenics were also produced carrying a WT LMNA BAC.
Sixteen founders who carry the transgene as determined by PCR
analysis were born and bred for the G608G constructs, eight from
H of the eight circular G608G BAC founders expressed progerin.
Offspring of founder H were used for the majority of studies
reported here. Of the eight linearized G608G BAC founders,
founders C, D, and H expressed progerin. For the WT BAC
transgenics, the circular construct produced five founders. Only
founder B expressed the transgene, and therefore, that line was
expanded. Of the three founders for the linearized WT BAC
construct, founder A expressed the transgene, and that line was
expanded. Southern blotting suggested a single copy of the BAC in
the G608G H-line, WT transgenic B-line, and WT transgenic
A-line mice (data not shown).
Consistent vascular pathologic changes were found in
founders F and H from the circular G608G BAC construct and
founder C from the linearized G608G BAC (see below).
Founder B from the circular WT BAC and founder A from the
linearized WT BAC showed no pathologic changes in any tissue,
except for possibly very mild fragmentation of elastin and
occasional small foci of proteoglycan (PG) deposition in the
medial layer of large vessels at advanced age.
RT-PCR and Western blotting indicated that the LMNA trans-
gene and the lamin A, lamin C, and progerin proteins were being
expressed in all tissues tested (bone, ear, skin, brain, heart, aorta,
sections of 12-month-old mice by using hematoxylin?eosin (H & E) and Movat’s
a progressive loss of VSMC, elastin breakage, thickened medial layer and adven-
titia, and PG accumulation. (B) All G608G mice examined over 16 months of age
showed evidence of carotid (shown) and aortic (data not shown) calcification,
mice showed no evidence of calcification. HA, hyaluronan.
transgenic construct by homologous recombination in
E. coli. Human BAC clone RP11-702H12 contains an
insert of 164.4 kb of genomic DNA from chromosome
1q including the LMNA gene. Homologous recombi-
nation was induced between this BAC and a shuttle
fragment of 2.3 kb containing the common G608G
kanamycin resistance gene (Kanr) flanked by two FRT
sites. Homology arms with sequences from intron 10
(I-10) and intron 11 (I-11) of the LMNA gene were used
fragment and the BAC clone. After selection for posi-
tive recombinants, Flpe recombinase was induced to
release the kanamycin gene. The final BAC G608G
transgenic construct used for pronuclear injection dif-
mutation (*) within exon 11 of LMNA and 109 extra
nucleotides in intron 10 including an FRT site, EcoRI
site, and SacI site.
Procedure for generating the BAC G608G
Varga et al.
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vol. 103 ?
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carotid artery, iliac artery, skeletal muscle, liver, kidney, spleen,
as supporting information on the PNAS web site, and data not
or skin quality were seen between the G608G H-line mice and the
WT transgenic and nontransgenic mice ?20 months of age.
consistent pathology was found in the external ear, skin, brain,
the G608G H-line mice. Sections of aorta, carotid artery, and iliac
artery were stained with hematoxylin?eosin and Movat’s penta-
chrome stains. In the G608G mice, the most dramatic finding was
the progressive loss of VSMC, elastic fiber breakage, thickening of
the adventitia and medial layer, accumulation of PGs, and collagen
deposition. This phenomenon was first observed in 5-month-old
mice and became severe by 12 months. Arterial calcification was
observed in older mice with severe VSMC loss and extracellular
matrix deposition. No inflammation was present, and the VSMC
that remained appeared hypertrophied. Although all large arteries
artery were the most severe. Affinity histochemistry confirmed an
accumulation of hyaluronan with age, which was absent in controls
(Fig. 2A). Von Kossa staining confirmed the progressive calcifica-
were absent in age matched controls (Fig. 2B).
To visualize expression of the transgene in microscopic sections,
we took advantage of the fact that the lamin A?C antibody
(Ab2559) recognizes both human and mouse lamin, but the lamin
not mouse lamin A. Immunohistochemistry using these antibodies
showed that in 5-week-old G608G and the controls lamin A?C was
in descending aorta by using either the mAb3211 antibody (detects human but not mouse) or the Ab2559 antibody (detects both human and mouse). The age
Cells with lamin A?C staining are present in the three layers of the arteries: intima, media, and adventitia in 5-week-old and 5-month-old transgenic mice. The
muscle ?-actin staining confirms loss of VSMCs; these images also show the eccentric distribution of vascular damage, with some areas around the vessel
circumference better preserved than others.
www.pnas.org?cgi?doi?10.1073?pnas.0600012103Varga et al.
present in the endothelial cells, in the medial VSMC, and in the
adventitia. Over the next 6–12 months, progressive loss of VSMC
was observed in G608G transgenics in an eccentric pattern around
(20 months), complete depletion of the medial VSMC occurred in
many regions (Fig. 3). Similar observations were made in the
carotid artery (see Fig. 7, which is published as supporting infor-
mation on the PNAS web site). We also noticed the presence in
lamin A but lack DNA (Fig. 3 and see Fig. 8, which is published as
supporting information on the PNAS web site). Smooth muscle
?-actin staining confirmed the eccentric VSMC loss in the medial
layer of large arteries, whereas the endothelial cells remained
essentially intact (Fig. 3 and see Fig. 7).
Transmission electron microscopy analysis of descending aorta
and carotid showed cellular debris, apparently derived from the
remnants of disintegrating VSMC. An abnormal extracellular
matrix was also visible, with accumulated PG, highly disorganized
collagen fibrils, and frayed elastic fibers displaying variable thick-
ness (Fig. 4).
The status of cell proliferation in the carotid artery and descend-
ing aorta was investigated by BrdUrd analysis on three mice from
H-line and control mice. Although the control tissue (thymus)
stained positive for proliferating cells, no cells were proliferating in
the vessel medial layer of transgenic and control mice (data not
shown). TUNEL staining of sections to look for evidence of
apoptosis was inconclusive.
In smooth muscle ?-actin knockout mice, blood pressure in
response to the vasodilator, sodium nitroprusside (NP), was ob-
served to be blunted (21). We, therefore, hypothesized that the loss
of VSMC in our transgenic mice would similarly lead to abnormal
arterial response to NP administration. Blood pressure was mea-
sured before and after NP infusion for G608G and nontransgenic
average baseline mean arterial pressure and blood pressure lower-
ing by NP was equivalent in the two groups. At 5 months of age,
baseline blood pressure tended to be slightly lower in the G608G
After infusion of NP, blood pressure lowering in 5-month-old mice
was significantly blunted in the G608G group, as measured by the
difference in mean arterial pressure before and after infusion (P ?
0.02, one-tailed t test). Values obtained at 5 months for the
difference in mean arterial pressure before and after infusion are
41, 38, and 40 mmHg (1 mmHg ? 133 Pa) for the nontransgenic
mice and 34 and 27 mmHg for the G608G mice. Additionally,
for several minutes after infusion in transgenic animals (Fig. 5).
To further the understanding of the pathogenesis of HGPS, we
created a mouse model by using the human LMNA gene
containing the G608G mutation. A knock-in might have initially
seemed like a more direct approach, but we were concerned that
introducing this single nucleotide change into the mouse Lmna
gene might or might not result in similar activation of a splice
donor and production of mouse progerin. Therefore, it seemed
more prudent to introduce the human G608G LMNA gene. We
chose to use a human BAC containing the LMNA gene and
substantial flanking DNA to include regulatory signals and
obtain a faithful developmental pattern of expression.
accumulation of cellular debris, frayed elastin fibers, and accumulation and
disorganization of collagen and PG (see black arrows). The nontransgenic
vessel shows only mild changes at 20 months. E, elastin; V, VSMC.
tative blood pressure tracings taken before and after NP administration in
5-month-old mice. (B) The drop in mean arterial pressure, comparing blood
pressure before and after the administration of NP, is blunted in the G608G
mice as compared with the control mice at 5 months of age.
Diminished vascular responsiveness in progeria mice. (A) Represen-
Varga et al.
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There were risks associated with this strategy. Creating an extra
copy of LMNA could cause a phenotype of its own. Regulation of
the human BAC transgene might be different in the murine
environment. Furthermore, the 164-kb BAC carries other genes
(UBQLN4, MAPBPIP, and RAB25) in addition to LMNA, which
could also affect the phenotype. All of these concerns were
except for possibly very mild medial arterial changes in animals of
advanced age, although this was difficult to distinguish from non-
G608G transgenic mice were found to have a progressive and
dramatic defect of the large arteries, consisting of progressive
medial VSMC loss and replacement with PG and collagen. In
the VSMC loss, with calcification and adventitial thickening. We
were unable to determine whether VSMC were undergoing apo-
ptosis or replicating to replace their loss because cell turnover was
too slow to detect with BrdUrd or TUNEL staining. These arterial
abnormalities were reflected functionally by an altered in vivo
response to the vasodilator NP. G608G mice demonstrated a
blunted initial response to NP, consistent with impaired vascular
relaxation and attenuated blood pressure recovery after infusion.
Although G608G transgenic mice lack pathologic features of
HGPS outside of the vascular system, the pathologic changes in
the medial layer of large vessels show pronounced similarity to the
human condition (2–5). Both human progeria patients and the
G608G transgenic mice have severe VSMC loss, accumulation of
acellular material, and calcification of the vessel wall. The pattern
in human progeria also includes some degree of intimal prolifera-
tion, which is also a form of vascular remodeling, probably in
response to the progressive loss of medial VSMC (2–5). We
observed minimal intimal thickening in the transgenic mice; how-
ever, it is possible that intimal thickening would occur in response
to vascular injury or other cardiovascular insults.
LMNA and progerin are expressed in a variety of tissues in these
transgenic animals. Why, then, is the mouse phenotype essentially
limited to VSMC in large arteries? Based on the report of Lam-
merding et al. (22) that lamin A?C-deficient mouse embryo fibro-
blasts are more sensitive to mechanical strain, we propose that
progerin-expressing VSMC in the aorta and proximal arterial tree
are in particularly vulnerable locations to experience mechanical
arterial walls, particularly in the aortic arch and at bifurcations,
progressively devitalizes VSMC that have been rendered unusually
fragile by the presence of progerin, ultimately leading to an almost
mice lived longer.
study potential therapies. For example, we and others recently
reported that the use of farnesyltransferase inhibitors can improve
the abnormal nuclear structure of human progeria fibroblasts in
23–25). Additionally, bone marrow transplantation from a com-
patible normal donor might provide stem cells that could repopu-
late the depleted arterial medial layer in HGPS. A similar strategy
has been reported to improve the clinical status of children with
osteogenesis imperfecta (26). Both drug therapy and transplant
approaches to progeria can now be tested with this mouse model.
Materials and Methods
Generation of LMNA G608G Transgenic Mice.ThehumanBACclone,
RP11-702H12 (RPCI-11 Human BAC Library, BACPAC Re-
source Center at Children’s Hospital Oakland Research Institute,
chromosome 1q including the LMNA gene (25.4 kb) and three
other known genes, UBQLN4, MAPBPIP, and RAB25. Recombi-
a shuttle fragment containing the G608G mutation, surrounding
upstream and downstream elements of the LMNA gene, and the
kanrgene flanked by FRT sites. The shuttle fragment was con-
structed by PCR amplification of genomic DNA from HGPS
sample AG11498 [carrying G608G (6)] with primers: NotI-
AACAGGGAACCCAGGTGTCT and EcoRI-AGGAAAAAT-
CATTCC and SalI-CAGGATTTGGAGACAAAGCAG; and by
PCR amplification of vector pIGCN21 with primers: EcoR1-
CGGGATCCACCGGATCTA and SacI-TGGAGGCTACCAT-
GGAGAAG. Following PCR amplification, the fragments were
Before recombineering, the shuttle fragment of 2.3 kb was released
from its carrier by digests with NotI and SalI and then was
gel-purified. Recombineering was performed in the recombino-
genic bacterial strain EL250 in accordance with procedures de-
scribed in refs. 20 and 27, and kanrclones were screened by PCR
for integration of the shuttle fragment by using primers: GTAGA-
CATGCTGTACAACCC and SacI-TGGAGGCTACCATG-
GAGAAG. Following identification of positive clones, FLP re-
(20). PCR of the different LMNA exons, sequencing of the region
targeted for recombination, and fingerprinting of BAC clone
digests with HindIII and XbaI from different steps in the recom-
bination process confirmed removal of the kanamycin resistance
gene and showed no additional recombination in the final clone
used for the production of the transgene. Double CsCl banding
BAC clones before injection into the male pronucleus of recently
fertilized C57BL?6 embryos. Unmodified purified RP11-702H12
was also injected for use as a control. Injections with circular and
linearized BAC clones were performed for each of the BAC clones
(RP11-702H12 G608G and RP11-702H12 unmodified). For lin-
earized injections, the CsCl-purified BAC DNA was digested with
PI-SceI, which cuts once in the vector, before injection. All animal
use complied with the Animal Care and Use Committee guidelines
(National Institutes of Health, Bethesda).
Mouse Genotyping. DNAwasextractedfromtailbiopsiesaccording
to standard phenol-chloroform methods. Genotyping was per-
formed on genomic DNA by PCR analysis by using primers to
amplify a fragment in intron 10 (5?-AACAGGGAACCCAGGT-
GTCT-3? and 5?-GCAGCAGGCATGCACTATTA-3?). The PCR
product for the G608G transgene is 545-bp long. The PCR product
for the WT transgene is 436-bp long. Southern blotting was also
genomic DNA and taking advantage of an EcoRI site that is
introduced into exon 10 in the G608G transgene. The probe was
lamin A cDNA to include intron 11 with BamHI and HindIII. The
1372-bp fragment that includes exons 6–11, intron 11, and exon 12
was cut from the 1% TAE gel and purified by using Gene Clean
a fragment of 9707 bp for hybridization to exons 6–10 from G608G
transgenic mice. Transgenic mice for the WT BAC were predicted
to have a hybridization band of 16,378 bp, and the probe binds a
10,406-bp fragment from endogenous mouse DNA.
Transgene Expression Analysis. RNA was extracted from homoge-
nized mouse tissues by using either TRIzol (Invitrogen) or the Fast
RNA Pro Green kit (Qbiogene). Reverse transcription was per-
formed by using Invitrogen’s SuperScript II RT-PCR kit. PCR of
the cDNA was performed by using primers 5?-GCAACAAGTC-
CAATGAGGACCA-3? and 5?-GTCCCAGATTACATGATGC-
3?. The PCR products produced include mouse lamin A (643 bp),
human lamin A (640 bp), and progerin (490 bp). To differentiate
www.pnas.org?cgi?doi?10.1073?pnas.0600012103Varga et al.
between endogenous mouse and transgenic human lamin A, BstUI Download full-text
was used to digest RT-PCR products, resulting in two bands for
human (386 and 254 bp) and one band for mouse (643 bp). Protein
Tris?glycine gels (Invitrogen) and probed with mAb3211 lamin A
antibody (Chemicon International) and goat anti-mouse horserad-
ish peroxidase secondary antibody (Kirkegaard & Perry Labora-
tories). Visualization of the bands was possible by using the
ECL-plus kit (Amersham Pharmacia Biosciences) and then devel-
oping the images on film (Kodak).
Histochemistry. Tissues fixed in 2% paraformaldehyde were dehy-
drated with graded alcohols and embedded in paraffin. Cross-
sections (4-?m thick) were cut with a rotary microtome and
mounted on charged slides (Superfrost, Columbia Diagnostics,
Springfield, VA) and then stained with hematoxylin?eosin and
an Olympus BX51 Microscope equipped with an Olympus DP11
digital camera. Images were managed by using Microsoft DIGITAL
IMAGE PRO 10 and Adobe PHOTOSHOP 7 for WINDOWS.
Immunohistochemistry. Staining for lamins was performed in PBS
by using the rabbit polyclonal anti-lamin A diluted 1:10 (Ab2559,
Abcam, Inc., Cambridge, MA) on transgenic or nontransgenic
tissues or the mouse monoclonal anti-lamin A?C nondiluted
goat anti-mouse Alexa Fluor 594-conjugated secondary antibodies
(Molecular Probes). Antigen retrieval was performed before the
incubation of the lamin antibodies and consisted of a 2-min
incubation in EDTA (0.37 g?liter) in a pressure cooker. A mouse-
to-mouse blocking kit (ScyTek Laboratories, Logan, Utah) was
used with mouse anti-lamin A?C. Staining for smooth muscle
?-actin was performed by using a Cy3-conjugated antibody diluted
1:100 (Sigma–Aldrich). Slides were mounted in DAPI-containing
obtained with a confocal microscope system (Zeiss LMS 510) and
collected with ?20 or ?40 oil lenses (Zeiss).
PGs were detected in paraffin sections of vessels, after digestion
with 0.2 units?ml chondroitin ABC lyase (Seikagaku America,
Rockville, MD) for 1 h at 37°C, by using rabbit anti-mouse
monoclonal antibodies (a gift from Larry Fisher, National Institute
Bethesda) against versican (LF99), biglycan (LF106), and decorin
perlecan (a gift from Koji Kimata, Nagoya University, Chikusa,
tor Laboratories) and visualized by using Vector Red reagent
(Vector Laboratories). Hyaluronan localization was carried out
with a commonly used biotinylated probe consisting of a mixture of
cartilage, PG, and link protein (provided by T.N.W.).
For BrdUrd analysis, the mice were given 100 mg?kg BrdUrd
(Sigma) via i.p. injection 18 and 1 h before death. Sections of aorta,
carotid, and thymus were prepared as discussed in ref. 29. Quan-
tification of proliferating cells was performed by three blinded
observers counting the number of BrdUrd-positive medial VSMC.
Systemic distribution of BrdUrd was confirmed by intense staining
of proliferating thymus cells in all animals receiving the agent.
Transmission Electron Microscopy. Samples were prepared and
(JEOL) as described in ref. 30. Images were acquired and saved on
Kodak 4489 electron microscope film with a ‘‘below the viewing
screen’’ film transfer system and scanned at 600 dots per inch by
using an Epson Expression 1680 flat bed scanner for digitization.
Ruthenium Red was used in some samples to gain better visual-
ization of PGs.
Blood Pressure Analysis. Blood pressure was measured invasively by
using a microtip pressure transducer catheter connected to an
electrostatic chart recorder (Millar Instruments, Houston). Ani-
carotid artery was cannulated. Once initial baseline blood pressure
measurements were completed, the mouse was infused with saline
nitroprusside (1.5 mg?kg of body weight, 30 ?l of total volume over
2 min) was then infused, and blood pressure measurements were
taken until a return to baseline or a maximum of 20 min. For
5-week-old mice, three G608G and three nontransgenic mice were
studied. For 5-month-old mice, two G608G and three nontrans-
genics were included. Because we postulated that the G608G
animals would have a blunted response to NP, we used a one-tailed
type-2 t test to assess significance, and this was performed by using
We thank Darryl Leja for assistance in preparing the figures,
Drs. David Bodine and Shelley Hoogstraten-Miller for mouse expertise,
Dr. Christian A. Combs and Daniela Malide [Light Microscopy Core
Facility, National Heart, Lung and Blood Institute, National Institutes of
Heath (NIH)] for assistance regarding microscopy-related experiments,
and Shih Queen Lee-Lin for DNA fingerprinting of BAC constructs.
This research was supported in part by the Intramural Research Program
of the National Human Genome Research Institute and the National
Heart, Lung, and Blood Institute (NIH) and by NIH Grant HL-18645 (to
T.N.W.). M.E. was supported by grants from the Tore Nilsson Founda-
tion, the Åke Wiberg Foundation, the Hagelen Foundation, the Loo and
Hans Osterman Foundation, the Torsten and Ragnar So ¨derberg Foun-
dation, the Jeansson Foundation, the Swedish Research Council, and the
Swedish Foundation for Strategic Research.
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