From the Departments of Pathology (M.D.R., N.S., R.D.S., C.W., J.-S.K.,
D.G.), Internal Medicine (R.D.S., G.J.N., E.G.N.), and Physiology (E.G.N.),
and Howard Hughes Medical Institute (G.J.N.), University of Michigan, Ann
Arbor.
Correspondence to David Gordon, MD, Vascular and Cardiac Diseases, Parke-Davis Pharmaceutical Research Division, 2800 Plymouth Rd, Ann Arbor, MI 48105. E-mail gordond{at}aa.wl.com
Methods and Results—In organ culture of rabbit aorta,
longitudinal stretch and supraphysiological
pressure applied for 3 hours during incubation with adenoviral vector
facilitated gene transfer into medial SMCs without denudation. Of the
SMCs, 10.2±3.8% expressed a reporter gene of human placental alkaline
phosphatase (hpAP), whereas SMCs in control arteries did not express
hpAP. To evaluate the feasibility of transgene expression in
arterial grafts, we performed such
permeabilization-assisted reporter gene transfer into aortas of donor
Dutch Belted rabbits and transplanted them into carotid arteries of
recipient New Zealand White rabbits. Unstretched transfected
grafts were used as a control. SMCs expressed hpAP (7.3±2.4% of cells
in 2 days and 4.2±1.9% in 2 weeks) in stretched grafts only. In the
next series of experiments, we transfected stretched grafts with
ADV-tk and combined transplantation with systemic
administration of ganciclovir. Stretched ADV-hpAP grafts were used as a
control. In 2 weeks, the formation of intimal thickening in
tk-expressing grafts was significantly reduced
(P<0.01) because of a decrease in proliferating
SMCs.
Conclusions—Manipulations within target tissues can enhance the
efficiency of gene transfer into SMCs. Although mechanical
permeabilization is clinically problematic, in principle,
targeting SMC replication may provide a genetic approach to the
treatment of transplant arteriosclerosis.
The tk gene, as well as the other genes being used to
inhibit in vivo SMC proliferation, encodes a protein with nuclear
localization, which normally cannot be secreted.8
Therefore, such therapy might work only if the transgene is expressed
in SMCs. However, most of the medial SMCs are resistant to gene
transfer unless the artery wall undergoes mechanical injury, such as
occurs with balloon angioplasty.9 10 11 12 At the same
time, preservation of vascular endothelium is required
for successful organ transplantation, and therefore, gene transfer into
medial SMCs should not entail deliberate endothelial
denudation.
We hypothesized that the endothelium and/or
extracellular connective tissue may limit viral vector penetration to
medial SMCs and that manipulations that increase the porosity of the
artery can make those cells prone to gene transfer. Accordingly, the
purpose of this study was 2-fold: to achieve transgene expression in
arterial SMCs without significant or irreversible
endothelial damage and to investigate whether the
proliferative response to allogenic arterial
transplantation can be inhibited by local expression of a tk
gene followed by ganciclovir administration.
Abdominal aortas (n=9) were obtained from intact New Zealand
White rabbits fed a regular rabbit chow. Before the animals were
euthanized, intravenous heparin was infused (300 U/kg). The
abdominal aorta was exposed, and both iliac and renal arteries were
cannulated. All other side branches were ligated. Through these ports,
the aorta was perfused in situ with culture medium and ligated. Then 2
ligatures were placed around the central part of the abdominal aorta
(distal to the renal arteries) to divide it into 2 halves. The ends of
the isolated segments were tied to surgical retractors (to prevent
longitudinal contraction of aortic tissue). Every segment was attached
to its own retractor. Later, the aorta was cut into 2 halves between
retractors. Both segments were then excised and transferred with the
retractors into separate Petri dishes containing culture media. To
increase the porosity of arterial wall, longitudinal
stretch was applied to the aortic segments. The retractors were spread
to 1.5 times the original length of aortic segment, and arteries were
kept stretched for 3 hours. ADV-hpAP solution (2 mL;
2x109 pfu/mL) was infused into the lumen via the
cannula, and the solution dwelled for 3 hours (dwell time) under a
pressure of 150 mm Hg. Control aortic segments were
pressurized and incubated with the vector as described above but did
not undergo longitudinal stretch. To control for the specificity of
gene expression, 3 aortas were transfected with ADV-
Transplantation of Transfected Rabbit Aorta
Diet
Gene Transfer
The group 2 experiment (Table 1
Transplantation surgery was performed similarly to the method of Bowyer
and Reidy.13 Recipient animals were
anesthetized with ketamine and xylazine as described
above and heparinized. A midline cervical skin incision was performed,
strap muscles were bluntly dissected, and both left and right common
carotid arteries were exposed. Aortic grafts were placed into both
carotid arteries of the recipient by a cuff anastomosis method (sterile
Teflon cuffs 4 mm long over which the arteries are stretched and
joined with a single circumferential suture).
Drug Treatment
Animal Euthanasia
Histochemistry
Triple-label immunocytochemistry was used to analyze
proliferation among different cell types by techniques that we have
described earlier in detail.15 To visualize
BrdU-positive cells, the cell proliferation kit (Amersham) was used.
The cell type–specific antibodies used on these tissues were (1) an
anti–smooth muscle
Scanning electron microscopy was used to evaluate intimal porosity in
the stretched arteries. Cultured aortic segments were perfused
with PBS and then with 2.5% glutaraldehyde. Specimens
were dehydrated and air-dried from hexamethyldisilazane (Sigma) and
sputter-coated with gold. To visualize subendothelial
connective tissue structures, fixed samples were incubated in potassium
ethoxide (30% KOH and 96% ethanol, 1:1) until
endothelial cells detached, as we described
previously.16
Morphometric Analysis
Measurements of intimal and medial cross-sectional area 14 days after
transplantation were determined by 2 independent reviewers in a blinded
fashion on 4 sections from each aortic graft with the image
analysis system. Intimal boundaries were determined by digital
planimetry. The entire circumference of neointima and media
was measured at x40 magnification. Average intimal and medial areas
were obtained from the 4 measurements per observer and then between the
2 observers. In addition, intima/media ratios were calculated for each
individual measurement and then averaged in the same fashion.
To evaluate proliferation of different cell types, the ratio of
double-labeled cells (BrdU-incorporated SMCs and macrophages)
to the total number of cells was calculated. Contiguous, nonoverlapping
microscopic fields covering the whole section were
analyzed.
Statistical Analysis
Most likely, luminal endothelium and/or underlying
connective tissue structures limited direct interaction between the
vector and the target SMCs. We hypothesized that longitudinal
mechanical stretch would induce the formation of transient holes
between endothelial cells as well as between any
connective tissue fibers, and intraluminal pressure would propel viral
particles through these holes. To test this hypothesis, aortic segments
were attached to surgical wound retractors, the retractors were spread
to 1.5 times the original length of the aortic segment, and arteries
were kept stretched for 3 hours. ADV-hpAP was infused into the lumen of
stretched segments under a pressure of 150 mm Hg. After the
incubation, the stretch was slowly released, and aortic tissue was cut,
maintained in culture, and fixed. Indeed, scanning electron microscopy
demonstrated the development of holes between luminal
endothelial cells (Figure 1C
Fate of Transgene-Expressing Cells in a Transplantation
Environment
Two days after transplantation, the pattern of hpAP expression was
reminiscent of that seen in organ culture. In both groups,
Expression of tk and Ganciclovir Administration
Inhibits Development of Transplant Arteriosclerosis
Fourteen days after transplantation, the thickness of the
neointima and intima/media ratio in
tk-transfected grafts were significantly reduced
(P<0.01, paired t test) compared with their
hpAP-transfected counterparts subjected to the same systemic factors
(Figures 3E
In the organ culture experiments, we found that medial SMCs of the
rabbit aorta did not express a transgenic reporter after incubation
with adenoviral vector. These data confirm previous in vivo
observations indicating that in normal arteries, gene transfer was
limited to luminal endothelium and
adventitia.9 10 11 It has also been demonstrated
that medial SMCs are able to express a transgene when adenoviral
particles are delivered either under very high pressure of 5
atm10 or after vigorous mechanical
injury6 12 permitting penetration of the vector.
However, the goal of this study was to inhibit SMC proliferation in
arterial transplants, and denudation of the graft before
transplantation would be counterproductive. We hypothesized that
transient graft permeabilization might facilitate gene transfer into
medial SMCs. To increase the porosity of the arterial wall,
we applied longitudinal mechanical stretch to the sealed segment of
aorta. The combination of longitudinal stretch with
supraphysiological intraluminal pressure allowed us
to perform efficient gene transfer into medial SMCs without
endothelial denudation.
It was unknown, however, whether these results were directly applicable
to the in vivo situation. Therefore, specific experiments were designed
to study the patterns of reporter gene expression in the aortic grafts
that had been transfected ex vivo and then transplanted into recipient
carotid arteries. We found that 2 days after transplantation, luminal
endothelial cells and various adventitial cells were
alkaline phosphatase–positive in both stretched and unstretched
grafts. However, only stretched grafts contained hpAP-expressing SMCs,
thus reproducing the pattern observed in organ culture. Fourteen days
after transplantation, comparable numbers of SMCs were still alkaline
phosphatase–positive, whereas hpAP gene expression was barely
detectable in the luminal endothelium and adventitia.
Disappearance of transgenic alkaline phosphatase activity might be
attributed to a cell-mediated immune response induced by
adenovirus-mediated gene transfer17 and/or
allogenic transplantation4 and is not surprising.
The apparently selective preservation of transgene expression by the
SMCs is more remarkable. The mechanisms of such selectivity are poorly
understood, because little is known about cell kinetics in this
extremely complex combination of allogenic transplantation,
immunosuppression, hypercholesterolemia, and
gene transfer. We think that the same anatomic barriers that did not
allow adenoviral particles to penetrate to aortic media could retard or
prevent the main participants of cell-mediated immune response (T cells
and macrophages) from gaining direct contact with at least the
majority of medial SMCs. As a result of such compartmentalization,
exposed "external" cells like endothelial and
adventitial cells could be preferentially killed while "hidden"
medial SMCs could be spared from the immune attack and maintain a
transgene expression, at least initially. Regardless of the mechanisms
of SMC protection, this phenomenon enabled us to proceed with gene
therapy experiments and, in particular, to determine a "therapeutic
window" for ganciclovir administration (day 7 to 14) when SMC
proliferation and transgene expression in SMCs coincided.
We realize that mechanical permeabilization of the arteries is far from
an ideal gene transfer–facilitating technique. Although complete
restoration of endothelial structural integrity was
demonstrated, it is possible that endothelium-dependent
responsiveness of the stretched vessels could be impaired. It is also
obvious that longitudinal stretch is clinically impractical for
solid-organ transplants, as opposed to the use of allograft artery
segments as bypass grafts. However, the important point is that medial
SMCs are not intrinsically resistant to gene transfer but
rather are hidden behind anatomic barriers. Our observations suggest
that any technique that would allow direct contact between the vector
and target SMCs would significantly increase the efficiency of gene
therapy. We hope that other more clinically relevant protocols will be
developed on the basis of this paradigm.
Currently, as a proof of the principle, we demonstrated that
stretch-facilitated tk gene expression and systemic
ganciclovir treatment reduced intimal formation in allogenic
arterial transplants. Our control experiments showed that
mechanical stretch and pressure per se did not stimulate intimal
growth. Mehra et al18 reported that
adenovirus-mediated reporter gene transfer also did not increase
intimal thickening in a similar rabbit model. These data suggest that
gene therapy targeting SMC proliferation may be used for a treatment of
transplant arteriosclerosis.
The effects on SMC proliferation are probably a result of inhibition of
DNA replication after phosphorylation of ganciclovir by
the enzyme thymidine kinase. The lethal effects of
phosphorylated ganciclovir probably are not limited to
transduced cells; inhibition of cell proliferation can be achieved by a
paracrine mechanism, the so-called "bystander
effect."8 We suggest that metabolic
cooperation may explain why in our experiments,
Recently, we applied the same treatment to an in vivo model of
restenosis in rabbit atherosclerotic
arteries.19 It was demonstrated that
tk-ganciclovir treatment of preexisting atherosclerotic
lesions significantly inhibited the proliferation of both SMCs and
macrophages after angioplasty. In the present study, the
overall reduction of neointimal thickening was accompanied
by an inhibition of SMCs but not macrophage proliferation. We
suggest that the major reason for such a disparity lies in the
difference between cellular targets in various experimental systems. In
preexisting atherosclerotic lesions, both SMCs and macrophages
were successfully transduced.19 In our
present experimental system, only SMCs were originally transduced
ex vivo, and macrophages infiltrated the artery later after
transplantation. Nevertheless, because SMCs but not macrophages
compose the bulk of intimal volume in transplant
arteriosclerotic lesions3 and
because SMCs, unlike macrophages, synthesize extracellular
matrix components, thus indirectly contributing to the growth of
neointima, inhibition of SMC proliferation is sufficient
for significant reduction of intimal thickness.
Thus, our results suggest that certain treatments can facilitate gene
transfer into elusive SMCs and that, in principle, targeting SMC
replication may provide a genetic approach to the treatment of
transplant arteriosclerosis.
Received January 21, 1998;
revision received April 7, 1998;
accepted April 16, 1998.
2.
Billingham ME. Cardiac transplant
atherosclerosis. Transplant Proc. 1987;19:19–25.[Medline]
[Order article via Infotrieve]
3.
Gordon D. Transplant
arteriosclerosis. In: Fuster V, Ross R, Topol EJ,
eds. Atherosclerosis and Coronary
Disease. Philadelphia, Pa: Lippincott-Raven Press; 1996:715–726.
4.
Libby P, Tanaka H. The pathogenesis of
coronary arteriosclerosis ("chronic
rejection") in transplanted hearts. Clin Transplant. 1994;8:313–318.[Medline]
[Order article via Infotrieve]
5.
Hayry P, Mennander A, Yilmaz S, Ustinov J, Raisanen A,
Miettinen A, Lautenschlager I, Lemstrom K, Bruggeman CA, Paavonen T.
Towards understanding the pathophysiology of chronic rejection.
Clin Invest. 1992;70:780–790.[Medline]
[Order article via Infotrieve]
6.
Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ,
Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell
proliferation after arterial injury. Science. 1994;265:781–784.
7.
Guzman RJ, Hirschowitz EA, Brody SL, Crystal RG,
Epstein SE, Finkel T. In vivo suppression of injury-induced vascular
smooth muscle cell accumulation using adenovirus-mediated transfer of
the herpes simplex virus thymidine kinase gene. Proc Natl Acad
Sci U S A. 1994;91:10732–10736.
8.
Borrelli E, Heyman R, Hsi M, Evans RM. Targeting of an
inducible toxic phenotype in animal cells. Proc Natl Acad
Sci U S A. 1988;85:7572–7576.
9.
Schulick AH, Dong G, Newman KD, Virmani R, Dichek DA.
Endothelium-specific in vivo gene transfer. Circ
Res. 1995;77:475–485.
10.
Rome JJ, Shayani V, Flugelman MY, Newman KD, Farb A,
Virmani R, Dichek DA. Anatomic barriers influence the distribution of
in vivo gene transfer into the arterial wall: modeling with
microscopic tracer particles and verification with a recombinant
adenoviral vector. Arterioscler Thromb. 1994;14:148–161.
11.
Lemarchand P, Jones M, Yamada I, Crystal RG. In vivo
gene transfer and expression in normal uninjured blood vessels using
replication-deficient recombinant adenovirus vectors. Circ
Res. 1993;72:1132–1138.
12.
Steg PG, Feldman LJ, Scoazec JY, Tahlil O, Barry JJ,
Boulechfar S, Ragot T, Isner JM, Perricaudet M. Arterial
gene transfer to rabbit endothelial and smooth muscle
cells using percutaneous delivery of an adenoviral
vector. Circulation. 1994;90:1648–1656.
13.
Bowyer DE, Reidy MA. Scanning electron-microscope
studies of the endothelium of aortic allografts in the
rabbit: morphological observations. J Pathol. 1977;123:237–243.[Medline]
[Order article via Infotrieve]
14.
Tanaka H, Swanson SJ, Sukhova G, Schoen FJ, Libby P.
Early proliferation of medial smooth muscle cells in coronary
arteries of rabbit cardiac allografts during immunosuppression with
cyclosporine A. Transplant Proc. 1995;27:2062–2065.[Medline]
[Order article via Infotrieve]
15.
Rekhter MD, Gordon D. Does platelet-derived growth
factor-A chain stimulate proliferation of arterial
mesenchymal cells in human atherosclerotic plaques? Circ
Res. 1994;75:410–417.
16.
Rekhter MD, Tertov VV, Andreeva ER, Kolpakov V, Mironov
AA, Orekhov AN. Lipid accumulation in the
subendothelial cells of human aortic intima impairs
cell-to-cell contacts: a comparative study in situ and in vitro.
Cardiovasc Pathol. 1993;2:53–62.
17.
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E,
Wilson JM. Cellular immunity to viral antigens limits E1-deleted
adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994;91:4407–4411.
18.
Mehra MR, Stapleton DD, Cook JL, Zhang T, Ventura HO,
Huang C, Maldonado B, Smart FW, Re RN, Murgo JP, Barbee RW.
Adenovirus-mediated in vivo gene transfer in a rabbit model of
allograft vasculopathy. J Heart Lung Transplant. 1996;15:51–57.[Medline]
[Order article via Infotrieve]
19.
Simari RD, San H, Rekhter M, Ohno T, Gordon D, Nabel
GJ, Nabel EG. Regulation of cellular proliferation and intimal
formation following balloon injury in atherosclerotic rabbit arteries.
J Clin Invest. 1996;98:225–235.[Medline]
[Order article via Infotrieve]
© 1998 American Heart Association, Inc.
Basic Science Reports
Graft Permeabilization Facilitates Gene Therapy of Transplant Arteriosclerosis in a Rabbit Model
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Smooth muscle cell (SMC)
replication plays a central role in the pathogenesis of transplant
arteriosclerosis. One strategy to eliminate
dividing cells is to express a herpesvirus thymidine kinase
(tk) gene that phosphorylates the
nucleoside analogue ganciclovir into a toxic form leading to cell
killing. However, medial SMCs are resistant to gene transfer
unless the artery undergoes deendothelialization. We
hypothesized that manipulations that increase the "porosity" of the
artery can make SMCs prone to gene transfer without
denudation.
Key Words: arteriosclerosis • genes • muscle, smooth • transplantation
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Transplant
arteriosclerosis is a thickening of the intima that
develops in the arteries of transplanted
organs.1 2 Smooth muscle cell (SMC) replication
plays a central role in its pathogenesis.3 4 5 One
strategy to selectively eliminate dividing cells is to express a
herpesvirus thymidine kinase (tk) gene, which
phosphorylates the nucleoside analogue ganciclovir into a
toxic form that leads to cell killing.6 7
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Recombinant Adenoviral Vectors
Three replication-deficient, recombinant adenoviral vectors were
constructed and purified as previously described. These vectors were
prepared from the adenovirus 5 serotype and contain deletions in the E1
and E3 regions, rendering them replication-deficient. The 3 adenoviral
vectors (ADV) included a vector encoding herpesvirus thymidine kinase
(ADV-tk), driven by a polyoma virus promoter and enhancer
and containing an SV40 polyadenylation sequence. An adenoviral vector
lacking a cDNA insert, ADV-
E1, was used for control experiments. A
third adenoviral vector, ADV-hpAP, encodes for a human placental
alkaline phosphatase (hpAP) reporter gene driven by a ß-actin
promoter and CMV enhancer with an SV40 nsfer in Organ Culture of
Rabbit Aorta E1. The aortic
segments were washed with media and cut into rings 3 to 4 mm
thick. Aortic rings were then transferred into 24-well plastic dishes,
maintained in the culture medium (medium 199, 10% FCS, 1% glutamine,
and 1% penicillin-streptomycin) for 2 days, and fixed in 10%
formalin.
Transplantation of aortic segments from Dutch Belted donors to
carotid arteries of New Zealand White rabbits was performed.
Cholesterol feeding was added for the recipient
rabbits (0.5% cholesterol diet supplemented with 2%
peanut oil, provided in 4-oz/d aliquots). It was started 1 week before
the transplantation and continued until euthanasia. Before surgery and
at euthanasia, serum cholesterol and
triglyceride levels were measured (Roche Biomedical
Laboratories).
Dutch Belted rabbits were anesthetized with
ketamine (35 mg/kg IM) and xylazine (5 mg/kg IM). The abdominal
aorta was harvested as described above. The group 1 experiments (Table 1
) were designed to study
the fate of the cells transfected with reporter gene in a
transplantation environment in vivo. To eliminate variability
potentially introduced by systemic factors, we harvested the abdominal
aorta from a donor rabbit, split it into 2 halves, and transfected 1
half with ADV-hpAP without longitudinal stretch (group 1A). The other
half was transfected with the same vector, but longitudinal mechanical
stretch was applied during incubation with adenovirus (group 1B). Then
these transfected grafts were transplanted into left and right common
carotid arteries, respectively, of the same recipient rabbit. The only
difference between the grafts was the presence or absence of
longitudinal stretch during incubation with ADV-hpAP.
View this table:
[in a new window]
Table 1. Design of Transplantation
Experiments 
) was designed to explore whether the
tk-ganciclovir treatment can inhibit the development of
transplant arteriosclerosis. The abdominal aorta of
a donor rabbit was attached to wound retractors and split into 2
halves. In short-term organ culture, the retractors were spread to 1.5
times the original length of aortic segment, and arteries were kept
stretched for 3 hours. One half of the aortic graft was
mock-transfected with ADV-hpAP (group 2A), and the other half was
infected with the ADV-tk (group 2B). Viral solution (2 mL;
2x109 pfu/mL) was infused into the lumen of the
stretched arteries at a pressure of 150 mm Hg.
Immune rejection of the aortic grafts (in both groups 1 and 2)
was suppressed by the administration of cyclosporine (5
mg · kg-1 ·
d-1 SC) beginning at the time of
transplantation. To inhibit cell proliferation in the transplant (group
2 only), ganciclovir (50 mg · kg-1
· d-1) was administered for 7 days beginning
at day 7 after the transplantation. This therapeutic window was chosen
because according to Tanaka et al14 and our own
pilot studies (data not shown), proliferation of medial SMCs in
arterial grafts starts in 1 week after the transplantation,
and in 2 weeks intimal thickening is detectable.
To analyze reporter gene expression in
transplanted arteries, animals of group 1 were euthanized by
administration of intravenous sodium pentobarbital (120 mg/kg) 2 days
and 14 days after surgery (Table 1). To assess the therapeutic
efficiency of a tk gene, animals of group 2 were euthanized
14 days after transplantation (Table 1). To label proliferating cells,
these rabbits received an intravenous infusion of
5-bromo-2'-deoxycytosine (BrdC, Sigma) 1 hour before
death. Aortic transplants were harvested, and each graft was cut into 6
rings 
3 mm wide. Rings 1, 3, and 5 were fixed in 10% formalin,
and rings 2, 4, and 6 were fixed in methacarn
(methanol:chloroform:acetic acid). Formalin-fixed material was used for
the histochemistry, and methacarn-fixed samples were collected for
immunocytochemistry.
Expression of recombinant hpAP protein was detected by
histochemical analysis. The slides were incubated in PBS at
65°C for 30 minutes to inactivate endogenous
alkaline phosphatase and then in PBS containing a
chromogenic substrate of 5-bromo-4-chloro-3-indolyl
phosphate-p-toluidine (1 mg/mL, Gibco BRL) and nitro blue
tetrazolium chloride (1 mg/mL, Gibco BRL) for 19 hours. This substrate
yields a dark purple–to-brown stain in the presence of alkaline
phosphatase. 
-actin antibody (Boehringer Mannheim) to
identify SMCs and (2) RAM11 (DAKO) to identify macrophages.
To evaluate the efficiency of gene transfer into rabbit aorta,
the specimens stained for alkaline phosphatase were studied by light
microscopy at x400 magnification with the Image 1 system of computer
image analysis (Universal Imaging Corp). Contiguous,
nonoverlapping microscopic fields covering the whole tissue on the
slide were analyzed. Each field was scored for the total number
of nuclei and the number of alkaline phosphatase–positive cells. The
average index per sample was then used to calculate the statistical
comparisons between different groups.
All values are expressed as mean±SEM. Average percentages of
hpAP-positive cells in each group of blood vessels in organ culture
were compared by 2-tailed, unpaired Student's t test.
Comparisons of percentages of hpAP-positive cells, as well as intimal
areas and BrdU labeling indices in aortic transplants, were made by
2-tailed, paired t test, because pairs of experimental and
control grafts were always obtained from the same donor and
transplanted into the same recipient.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Optimization of Gene Transfer Into Normal Rabbit Aorta in
Organ Culture
The alkaline phosphatase histochemical reaction showed no staining
in the mock-transfected (ADV-E1) aortas (Figure 1A). Staining of the specimens incubated
with ADV-hpAP for 3 hours revealed that all endothelial
cells and variable numbers of adventitial cells expressed hpAP.
However, no medial SMCs (located centrally) showed alkaline phosphatase
positivity (Figures 1B and 2).
View larger version (144K):
[in a new window]
Figure 1. Optimization of gene transfer conditions in organ
culture of rabbit aorta. A, Alkaline phosphatase histochemical reaction
shows no staining in mock-transfected (ADV- E1, 2x109
pfu/mL for 3 hours) rabbit aorta, thus demonstrating specificity of
assay. B, Pattern of hpAP expression in organ culture of normal aorta
incubated with ADV-hpAP at optimal titer (2x109 pfu/mL)
for 3 hours. Luminal endothelial cells and adventitial
cells express transgenic hpAP (dark product of histochemical
reaction, arrows). C, Normal rabbit aorta in organ culture. Scanning
electron microscopy of luminal surface shows tightly packed,
overlapping endothelial cells. Magnification x1500. D,
Longitudinally stretched segment of rabbit aorta. Scanning electron
microscopy reveals formation of gaps between luminal
endothelial cells (arrows). Magnification x1900. E,
Tight meshwork of connective tissue fibers in
subendothelium of normal rabbit aorta in organ culture.
Scanning electron microscopy, magnification x400. F, Formation of wide
gaps (*) between subendothelial connective tissue
fibers in longitudinally stretched rabbit aorta. Scanning electron
microscopy, magnification x400. G, Luminal endothelium
restores its integrity after release of stretch and pressure. Scanning
electron microscopy, magnification x1500. H, Pattern of transgene hpAP
expression in organ culture of rabbit aorta incubated with ADV-hpAP for
3 hours under stretch and pressure. Luminal endothelial
cells and adventitial cells express transgenic hpAP (dark product
of histochemical reaction, arrows), as in intact aortas. In addition,
medial SMCs show reporter gene expression (arrowheads). Alkaline
phosphatase histochemical reaction. Methyl green nuclear counterstain.
Objective x20. L indicates lumen.
View larger version (15K):
[in a new window]
Figure 2. Percentage of hpAP-expressing medial SMCs in
longitudinally stretched and pressurized vs untreated rabbit aortas, 2
days in organ culture. and 1D) and between
extracellular connective tissue fibers (Figure 1E and 1F). Normal
morphology was restored after the release of mechanical stretch (Figure 1G). Corresponding to these acute alterations of vascular structure, we
found that 2 days after stretch-assisted gene transfer, 10.2±3.8% of
medial SMCs expressed the transgene (Figures 1H and 2). Thus, it was
concluded that mechanically enhanced porosity of the
arterial wall facilitated gene transfer into medial SMCs
without endothelial denudation. This
stretch-and-pressure approach was subsequently used in our in vivo
transplantation experiments.
To analyze reporter gene expression over time after
transplantation, we performed ex vivo hpAP gene transfer into aortas
derived from Dutch Belted rabbits and transplanted them into the
carotid artery position of recipient New Zealand White rabbits. Control
grafts were infected without longitudinal stretch (group 1A), whereas
experimental grafts underwent mechanical treatment during incubation
with ADV-hpAP (group 1B). 100% of
luminal endothelial cells and 22% of adventitial
cells were alkaline phosphatase–positive (Figure 3A and 3B, Table 2). In the control group,
there was no transgene expression in medial SMCs, whereas 7.3±2.4% of
medial SMCs in previously stretched grafts demonstrated recombinant
protein activity (Figure 3A and 3B, Table 2). Two weeks after surgery,
transplantation-associated intimal thickening had developed in both
stretched and unstretched grafts (intimal area, 1.2±0.2 and
1.1±0.3 mm2, respectively,
P=NS). These values were not different from a baseline group
that was transplanted but not subjected to gene transfer (1.0±0.3
mm2). Almost no endothelial cells
covering intimal thickening or adventitial cells expressed the
transgene in either group (Figure 3C and 3D, Table 2). However,
4.2±1.9% of SMCs in the experimental group demonstrated persistent
hpAP expression (Figure 3D, Table 2). These cells were located both in
the graft media and, less frequently, in the intima. Thus, ex
vivo–transduced SMCs, unlike endothelial cells and
fibroblasts, are capable of relatively long-term transgene expression
in a transplantation environment in vivo. Longitudinal mechanical
stretch and pressure applied during the gene transfer procedure
facilitated transgene expression in SMCs without additional stimulation
of intimal thickening.
View larger version (87K):
[in a new window]
Figure 3. Gene transfer and transplant
arteriosclerosis. A and B, Pattern of transgenic
hpAP expression in rabbit aortic allografts 2 days after
transplantation. Grafts were incubated with ADV-hpAP for 3 hours ex
vivo before transplantation, without (A) and with (B) longitudinal
stretch. Recipient rabbits underwent cholesterol feeding
and cyclosporine treatment. In both cases, all luminal
endothelial cells and some adventitial cells express
transgenic hpAP (purple product of histochemical reaction, arrows).
In addition, medial SMCs in a previously stretched graft (B) show
reporter gene expression (arrowheads). L indicates lumen. Alkaline
phosphatase histochemical reaction. Methyl green nuclear counterstain.
Objective x20. C and D, Pattern of transgenic hpAP expression in
rabbit aortic allografts 14 days after transplantation. Grafts were
incubated with ADV-hpAP for 3 hours ex vivo before transplantation,
without (C) and with (D) longitudinal stretch. Recipient rabbits
underwent cholesterol feeding and cyclosporine
treatment. In both cases, luminal endothelial cells and
adventitial cells do not express transgenic hpAP. However, medial SMCs
in a previously stretched graft (D) maintain reporter gene expression
(arrowheads). Alkaline phosphatase histochemical reaction. Methyl green
nuclear counterstain. Objective x20. E and F, Inhibition of intimal
thickening in rabbit aortic allografts by
ADV-tk/+ganciclovir 14 days after transplantation.
Control graft (E) was infected with ADV-hpAP and experimental graft (F)
with ADV-tk before transplantation (both were stretched and
pressurized during incubation with vector). Recipient rabbits underwent
cholesterol feeding, cyclosporine, and
ganciclovir treatment. Arrows indicate internal elastic lamina.
Hematoxylin and eosin staining. Objective x20. G and H, Proliferation
of different cell types in rabbit aortic allografts, 14 days after
transplantation. Control graft (G) was infected with ADV-hpAP, and
experimental graft (H) with ADV-tk before transplantation
(both were stretched and pressurized during incubation with vector).
Recipient rabbits underwent cholesterol feeding and
cyclosporine and ganciclovir treatment. Triple
immunolabeling for BrdU (black product of peroxidase
reaction), smooth muscle–specific -actin (red product of
alkaline phosphatase reaction), and macrophage-specific
antigen RAM11 (blue product of alkaline phosphatase reaction)
demonstrates predominantly proliferating SMCs (black nuclei colocalized
with red cytoplasm, arrows) in ADV-hpAP infected grafts (G), and
predominantly proliferating macrophages (black nuclei
associated with blue cytoplasm, arrowheads) in ADV-tk
infected grafts (H). Methyl green nuclear counterstain. Objective
x100.
View this table:
[in a new window]
Table 2. Percentage of hpAP-Expressing Cells in Rabbit Aortic
Grafts
To explore whether the cytotoxic effects of
tk-ganciclovir treatment can inhibit the development of
transplant arteriosclerosis, we obtained abdominal
aortas from Dutch Belted rabbits, split each aorta into halves, and
stretched and pressurized both of them to ensure effective vector
delivery to the target SMCs. In each pair, 1 segment was infected with
ADV-hpAP (control, group 2A) and another segment with ADV-tk
(group 2B). Every pair of grafts was then transplanted into carotid
arteries of New Zealand White rabbits, as described for group 1.
Recipient animals were kept on a cholesterol diet (started
a week before surgery) and received cyclosporine (started
immediately after surgery and continued until euthanasia) exactly as
described for the group 1 experiments. In addition, recipient animals
in the group 2 experiment received daily injections of ganciclovir for
7 days (days 7 through 14 after transplantation). , 3F, 4A, and 4B). This
reduction in neointimal area was accompanied by a
significant decrease in SMC proliferation, measured by triple-label
studies of BrdU, -actin, and RAM11 (Figure 3G and 3H). SMC
proliferation, expressed as a ratio of BrdU-positive,
-actin–positive cells to the total cell number (intima + media)
dropped from 2.3±0.2% in hpAP-expressing (control) grafts to
1.1±0.2% in tk-expressing (experimental) grafts
(P<0.01, paired t test). In this animal model, a
decrease in macrophage proliferation was not observed (Figure 3G and 3H). Moreover, the percentage of BrdU-positive, RAM11-positive
cells in tk-expressing grafts increased (2.5±0.9%)
compared with hpAP-expressing grafts (1.0±0.3%; P<0.01,
paired t test). However, triple-label studies suggest that
SMCs, rather than macrophages, contribute to the majority of
intimal volume (Figure 3G and 3H).
View larger version (25K):
[in a new window]
Figure 4. Effects of ADV-tk/+ganciclovir on
intimal thickening 14 days after transplantation. Control grafts were
infected with ADV-hpAP, and experimental grafts with ADV-tk
before transplantation (both were stretched and pressurized during
incubation with vector). Recipient rabbits underwent
cholesterol feeding and cyclosporine and
ganciclovir treatment. Each recipient hosted 2 grafts (1 infected with
ADV-hpAP and 1 infected with ADV-tk). Data are
presented as matched pairs of samples (control and experimental
grafts), whereby each pair belongs to same recipient. Each point in
chart represents average intimal area of individual graft.
Connected points represent pairs of grafts transplanted into
same recipient animal. Points on left show average intimal areas of
ADV-hpAP transfected grafts, and points on right, average intimal areas
of ADV-tk transfected grafts. A, Intimal area. Two-tailed,
paired t test: P<0.01. B, Intima/media
ratio. Two-tailed, paired t test:
P<0.01.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Gene therapy is a reasonable approach to transplant
arteriosclerosis, because the disease is limited to
graft arteries and the graft itself is available for ex vivo
manipulations before transplantation. SMC replication plays a central
role in the pathogenesis of transplant
arteriosclerosis.3 4 5 To
design rational gene therapy targeting SMC proliferation, 3 major
questions should be addressed: (1) How should a vector be delivered
specifically to target medial SMCs? (2) What is the fate of transduced
cells in the transplantation environment? (3) Does gene therapy inhibit
SMC proliferation, and does this result in a reduction of graft intimal
thickening? 50% reduction of
intimal thickness was observed but only 10% of SMCs were originally
transduced. It is unclear whether the bystander effect is functional
between dissimilar cell types like SMCs and macrophages.
Acknowledgments
This study was supported in part by grants from the Michigan
Affiliate of the American Heart Association (Drs Gordon and Rekhter)
and from the National Institutes of Health (HL-43757 to Dr E.G. Nabel).
Dr E.G. Nabel is an Established Investigator of the American Heart
Association. We gratefully acknowledge Dawn Lutz and Dr Val Kolpakov
for technical expertise.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Weis M, von Schneidt W. Cardiac allograft
vasculopathy: a review. Circulation. 1997;96:2069–2077.
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