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(Circulation. 2000;102:2629.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Rapid and Efficient Vascular Transport of Arginine Polymers Inhibits Myointimal Hyperplasia
From the Department of Medicine, Stanford University School of Medicine, Stanford, and CellGate Inc (J.B.R.), Sunnyvale, Calif.
Correspondence to John P. Cooke, MD, PhD, Associate Professor and Director, Section of Vascular Medicine, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305-5246. E-mail John.Cooke{at}Stanford.edu
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Abstract |
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Background—We recently discovered that short polymers of arginine efficiently translocate across the cytoplasmic membrane independent of the basic amino acid transporter. We evaluated the kinetics and biological effects of heptamers of L-arginine and D-arginine (L-R7 and D-R7, respectively) in vascular cells. We assessed the effects of these peptides on the NO synthesis pathway and vascular cell proliferation.
Methods and Results—Human umbilical vein endothelial cell and rabbit vascular segments were incubated in medium containing biotin-labeled L-R7 or D-R7. Both polymers rapidly translocated through the vessel wall and into the vascular cells in a dose- and time-dependent fashion. At a dose of 10 µmol/L for 30 minutes, 100% of the endothelial cells showed evidence of cytoplasmic and nuclear localization of the peptides. To evaluate the biological effects of the polymer translocation on myointimal formation, rabbit jugular vein segments were incubated with polymers (10 µmol/L, 30 minutes) or vehicle before arterial interposition grafting. Planimetric measurement 28 days after surgery revealed that L-R7 and D-R7 substantially reduced myointimal formation compared with the control condition (intima/media ratio: control 1.50.5, L-R7 0.40.2, and D-R7 0.80.2; P<0.05). Furthermore, basal nitrate and nitrite production from L-R7–treated grafts was significantly higher than that from both control and D-R7–treated veins. Studies in vitro of cultured vascular smooth muscle cells revealed that both polymers also exhibit an NO-independent inhibition of vascular smooth muscle cell proliferation.
Conclusions—Short polymers of arginine have the unique ability of vascular cell translocation, and they also have direct biological effects. These attributes are potentially useful in treating myointimal hyperplasia.
Key Words: amino acids • nitric oxide • muscle, smooth
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Introduction |
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Myointimal hyperplasia is a vascular response to injury that contributes to the development of vein graft disease, restenosis after angioplasty, and atherosclerosis.1 2 Myointimal hyperplasia involves the migration and proliferation of vascular smooth muscle cells (VSMCs) as well as the elaboration of extracellular matrix in the intima.3 4 Vascular NO inhibits myointimal formation by suppressing monocyte adherence and infiltration and VSMC proliferation and by inducing VSMC apoptosis.5 6 7 8 Systemic or local supplementation of L-arginine, an NO synthase substrate, enhances vascular NO production and inhibits vascular myointimal formation.9 10 11 12 13 Ordinarily, NO synthesis is not substrate-limited. However, under certain circumstances, L-arginine can become rate-limiting, as with elevated plasma levels of asymmetric dimethylarginine, a circulating NO synthase antagonist,14 15 or with the expression of the inducible NO synthase (iNOS).16 17 18 Both of these abnormalities are operative in the setting of vascular injury.19 20 21 Furthermore, in some pathological conditions, impairment of L-arginine uptake occurs through its membrane transporters.22 23 Under these circumstances, the efficient intracellular delivery of L-arginine may attenuate myointimal hyperplasia.
We recently have observed that short polymers of between 6 and 15 residues of arginine, but not lysine or histidine, efficiently translocate across the cytoplasmic membrane of cultured Jurkat cells independently of the membrane basic amino acid transporter.24 In addition to their role as intracellular transporters, polymers of L-arginine might be useful as an intracellular source of L-arginine, which in vascular tissue might result in therapeutically useful NO release. To test this hypothesis, we evaluated the kinetics of heptamers of L-arginine and D-arginine (L-R7 and D-R7, respectively) in vascular cells and tissue and also the biological effects of these polymers on myointimal hyperplasia in a vein graft model.
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Methods |
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Peptide Synthesis
Peptides were synthesized by using solid-phase techniques and Fmoc amino acids, resins, and reagents (PE Biosystems and Bachem) on an Applied Biosystems 433 peptide synthesizer.25 The reagents used in the present study were (1) L-R7 (NH2-RRRRRRR-COOH, R=L-arginine), (2) D-R7 (NH2-rrrrrrr-COOH, r=D-arginine), (3) L-R5 (NH2-RRRRR-COOH), (4) D-R5 (NH2-rrrrr-COOH), (5) biotin-labeled L-R7 (bL-R7, biotin-Aca-RRRRRRR-CONH2), (6) biotin-labeled D-R7 (bD-R7, biotin-Aca-rrrrrrr-CONH2), and (7) biotin-labeled L-K7 (bL-K7; biotin-Aca-KKKKKKK-CONH2, K=L-lysine).
In Vitro Translocation Study
Spontaneously transformed human umbilical vein
endothelial cells (ECV304, American Type Culture
Collection) were cultured in medium M199 (Irvine Scientific) containing
10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin
(GIBCO-BRL). Confluent cells were washed and placed in serum-free
medium. After 2 hours, the cells were incubated in the presence of
biotin-labeled peptides, such as bL-R7, bD-R7, or bL-K7 (0.1, 1.0, and
10.0 µmol/L). To assess the role of endocytosis in the cellular
uptake of the peptides, experiments were performed at 4°C. To
determine whether the uptake of R7 was an energy-requiring process,
some experiments were carried out in the presence of sodium azide
(1.0%) to deplete the cells of ATP and GTP. The cells were exposed to
sodium azide for 30 minutes before addition of the peptides. After 30
minutes of incubation, cells were stained with trypan blue to assess
cell viability or were washed 3 times with PBS, fixed in
ethanol/acetone, washed in PBS, incubated for 30 minutes with
peroxidase suppressor (ImmunoPure, Pierce) to block
endogenous peroxidase activity, and then incubated with 5
µg/mL horseradish peroxidase–conjugated streptavidin (Pierce) for 30
minutes. A substrate of HRP, diaminobenzidine (DAB, Sigma Chemical Co),
was added to the cells. The reaction was terminated by washing in
distilled water after a 60-second incubation.
Ex Vivo Translocation Study
Carotid artery and jugular vein segments of male New Zealand
White rabbits were used. To test the dose dependency of R7
translocation, vascular segments were incubated for 30 minutes with
either bL-R7 or bD-R7 solution (0.1, 1.0, and 10.0 µmol/L) in
serum-free DMEM (GIBCO-BRL). To test the incubation time dependence,
vascular segments were incubated with 10.0 µmol/L biotin-labeled
R7 (bR7) solution for 10 seconds, 60 seconds, 5 minutes, 10 minutes,
and 30 minutes. To determine the ability of R7 to penetrate through the
vessel wall, R7-containing medium was instilled into the lumen, and the
vessel was ligated proximally and distally to expose only the luminal
surface to R7 (10.0 µmol/L) for 30 minutes. To test the
temperature dependence of translocation, vascular segments were
incubated with 10.0 µmol/L bR7 solutions at 37°C or 4°C. To
determine the disappearance time course of translocated R7, vascular
segments were incubated with bR7 solutions (10.0 µmol/L) at
37°C for 30 minutes and then reincubated in DMEM with 10% FBS up to
5 days. Vascular segments were harvested 1, 2, and 5 days later.
Histological Detection of Internalized bR7
After incubation with bR7, vascular segments were frozen in OCT
compound (Miles Scientific). Frozen sections, 5 µm thick, were
fixed with acetone for 10 minutes. Internalized biotin was detected by
using the staining procedure described above. Methyl green was used for
nuclear counterstaining.
To quantify the efficiency of R7 nuclear translocation, the numbers of both DAB-positive nuclei and total nuclei were counted in the intima and media separately at x400 magnification with a video image analysis system (Automatrix). The frequency of R7 nuclear translocation was expressed as the percent staining of nuclei, defined as the ratio of the number of DAB-positive nuclei to that of all nuclei, in the intima and media.
Surgical Procedure
Male New Zealand White rabbits (3.0 to 3.5 kg) were
anesthetized with a mixture of ketamine (40 mg/kg) and
xylazine (5 mg/kg) intramuscularly. The left external jugular vein was
excised and immersed in PBS (control) or PBS containing either L-R7
(10.0 µmol/L) or D-R7 (10.0 µmol/L) for 30 minutes. As
controls, L-R5 and D-R5 (10.0 µmol/L) were also used, which in
Jurkat cells do not translocate across the cell
membrane.24 The right common carotid artery was exposed
and clamped at the proximal and distal ends. The treated vein segment
was anastomosed in a reverse end-to-side fashion into the carotid
artery with use of continuous 8-0 polypropylene sutures. The common
carotid artery was ligated and dissected between the 2 anastomoses, and
the wound was closed with 3-0 nylon suture. The experimental protocols
were approved by the Administrative Panel on Laboratory Animal Care of
Stanford University.
Vessel Morphometry
Vein graft segments were harvested on the 28th surgical day.
Graft segments were fixed in 10% buffered formalin with gentle
intraluminal pressure. The middle portions of the paraffin samples were
sectioned (5 µm) and stained with hematoxylin-eosin. Three
sections of each graft, taken at 0.5-mm intervals, were
analyzed by planimetry by a observer blinded to the treatment
group. The cross-sectional areas of the lumen, intima, and media were
digitized by use of the Image Analyst program (Automatrix). The
intima-to-media (I/M) area ratio was calculated.
Measurement of Ex Vivo NOx Production From Vein
Grafts
Vein grafts were harvested 3 days after surgery. Vein graft
segments were incubated in 1 mL Hanks’ buffered saline solution (HBSS,
Irvine Scientific) containing calcium (1.0 mmol/L) and
L-arginine (100 µmol/L, Sigma) at 37°C for 2
hours. Nitrate and nitrite (NOx) production was measured either
in the absence (basal) or presence (stimulated) of calcium ionophore
(A23187, 10.0 µmol/L, Sigma). Samples of the medium (80 µL)
were collected, and NOx measurement was performed by using the Griess
reaction and a commercial colorimetric assay (Cayman
Chemical).
VSMC Proliferation Assay
Rat aortic VSMCs were grown to 50% confluence in 96-well cell
culture plates. VSMCs were incubated with serum-free DMEM for 48 hours
to obtain quiescent cells. Western analysis confirmed the
absence of iNOS in these cells. Thereafter, VSMCs were treated with
vehicle, L-R7 (10.0 µmol/L), or D-R7 (10.0 µmol/L) for 30
minutes. After the treatment, cells were washed and incubated for 48
hours with serum-containing DMEM (0.5%, FBS). Cell count was performed
by use of a proliferation assay kit with spectrophotometry (XTT,
Boehringer-Mannheim). As a negative control, cells treated with
vehicle and incubated with serum-free medium were used. As an index of
cell proliferation, the optical density ratio of each treatment group
to the negative control group was calculated as an index of cell
proliferation.
Statistical Analysis
All values in text are expressed as mean±SEM. Means were
compared by ANOVA, and a value of P<0.05 was accepted as
statistically significant.
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Results |
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In Vitro Translocation of R7
No internalized biotin was detected by light microscopy in control cultures exposed to either vehicle or bL-K7 (Figure 1A
and 1B). On the other hand, even at
the lowest concentration of b-R7 (0.1 µmol/L), internalized
biotin was observed in the cytoplasm of all endothelial
cells (Figure 1C). At 10.0 µmol/L b-R7 for 30 minutes,
internalized biotin was also detected in the nucleus of virtually all
exposed cells, with accumulation in the nucleoli (Figure 1D).
There were no observable differences in the distribution or intensity
of internalized biotin between L-R7 and D-R7. This transport was an
energy-requiring process, because when rat vascular smooth muscle cells
were exposed to 1% sodium azide for 30 minutes before incubation with
bR7, neither cytoplasmic nor nuclear staining was observed. Trypan blue
staining indicated that these concentrations of the arginine polymers
did not induce cytotoxicity. These findings indicate that both L-R7 and
D-R7 are very efficient at translocating across both cytoplasmic and
nuclear membranes of endothelial cells in culture and
act as carriers for a second molecule, biotin.
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Ex Vivo Translocation of R7
When the carotid artery and jugular vein were incubated with bL-K7
(10 µmol/L), no uptake of biotin was apparent (Figure 2A and 2B). On the other hand, after
incubation for 30 minutes at a dose of 10.0 µmol/L bL-R7, a
distinct biotin signal was observed in virtually all intimal cells,
medial cells, and adventitial cells (Figure 2C and 2D and Figure 3A). Jugular vein segments, incubated
with bL-R7, exhibited a similar staining pattern (Figure 2E).
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The extent and the intensity of staining increased in a time-dependent
manner, so that within 30 minutes, virtually all vascular cells
exhibited a distinct biotin signal in both the cytoplasm and nucleus
(Figure 3B
and 3C).
Furthermore, when b-R7 was instilled intraluminally, biotin signals
were detected even in the adventitial cells, which stained intensely
after intraluminal exposure for 30 minutes (Figure 2F). There
were no differences between D-R7 and L-R7 in their ability to penetrate
the vascular wall and translocate into cells. Intriguingly,
translocation of bL-R7 into vascular tissue occurred when the
experiments were performed at 4°C, indicating that R7 translocation
was not dependent on classic endocytosis (Figure 3B and 3C).
To estimate the relative stability of D-R7 and L-R7 in vivo, the
disappearance of the biotin signal over time from vascular segments ex
vivo was studied. Residual nuclear biotin in both
endothelial and medial cells was greater in vascular
segments treated with bD-R7 at days 1 and 2 after exposure (Figure 2G and 2H). No significant positive staining was observed with
either form of R7 by day 5 (Figure 4A and 4B).
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Biological Effects of R7 on Myointimal Formation of Vein
Grafts
All vein grafts treated with vehicle developed significant
myointimal hyperplasia 28 days after surgery (Figure 5). By contrast, vessel segments treated
with L-R7 or D-R7 had substantially less myointimal formation (intimal
area: control 1.70.8 mm2, L-R7 0.50.2
mm2, and D-R7 1.10.4
mm2; P<0.05). Treatment was more
potent with L-R7 than with D-R7, and L-R7 treatment reduced the intimal
area by >70%. The I/M ratio of L-R7–treated vein grafts was also
significantly less than that of both control and D-R7–treated grafts
(I/M ratio: control 1.50.5, L-R7 0.40.2, and D-R7 0.80.2;
P<0.05) (Figure 6). Treatment
using the smaller oligopeptide (eg, R5) did not inhibit myointimal
formation.
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Mechanisms by Which R7 Inhibits Myointimal Hyperplasia
Three days after surgery, the basal NOx production from
L-R7–treated vein grafts was significantly higher than that from both
control and D-R7–treated vein grafts (NOx production: control
356 nmol/L per milligram tissue per hour, L-R7 8014 nmol/L per
milligram tissue per hour, and D-R7 488 nmol/L per milligram tissue per
hour; P<0.05). There was no significant difference in basal
NOx production between D-R7 and vehicle-treated vein grafts.
Calcium ionophore stimulation of eNOS did not affect NOx
production by the vein grafts (Figure 7A).
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Because D-R7 significantly reduced myointimal formation (although
to a lesser degree than L-R7) without enhancing NO production,
we evaluated whether arginine polymers had direct cytostatic effects.
Indeed, cell proliferation assays revealed that (in the absence of NOS
enzyme) VSMC proliferation was significantly inhibited by pretreatment
with both L-R7 and D-R7 compared with vehicle incubation. There were no
significant differences between isomer treatment groups in this
NO-independent cytostatic effect (Figure 7B).
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Discussion |
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The salient observations of the present study are as follows: (1) Arginine heptamers (but not pentamers) are extraordinarily efficient at translocating through the vessel wall and into the cytoplasm and nucleus of vascular cells. (2) Arginine heptamers suppress vascular smooth muscle cell proliferation and reduce myointimal hyperplasia in vein grafts by NO-dependent and -independent pathways. These observations are significant because they may herald a new and effective approach for local delivery of vascular therapeutics. The unique characteristics of R7 to rapidly translocate across the cytoplasmic and nuclear membrane of vascular cells may make it an excellent candidate as a carrier for vascular therapeutics.
Recently, we observed that polymers of arginine, but not lysine or histidine, efficiently enter cultured cells in vitro.24 In the present study, we found that heptamers of arginine (R7) translocated into cultured vascular cells and vascular tissue in a dose- and time- dependent manner. Moreover, when applied intraluminally, R7 was capable of penetrating deeply into the vessel wall. The ability to cross biological membranes is not due to the polycationic character of the peptide but rather to the guanidinium groups of arginine, because K7 did not enter the cells effectively. Furthermore, when biotin was linked to the peptides, it was delivered into the vessel wall. Peptides composed of D-arginine are more resistant to hydrolysis than are those composed of L-arginine, and this property may be advantageous for certain forms of drug delivery.
The cellular uptake of R7 is an energy-dependent process, because sodium azide blocked the uptake and nuclear localization of the peptide. However, R7 translocated into the cells even at 4°C, which suggests that conventional endocytotic pathways do not play an important role in R7 translocation.26 Once in the cell, R7 also crossed the nuclear membrane. In the lymphocyte cell line, the Tat protein of HIV-1 efficiency translocates across cytoplasmic and nuclear membranes. It has been suggested that the short basic region of Tat, which is composed of an arginine-rich sequence (residues 49 to 57, RKKRRQRRR), is responsible for this unique property.27 28 29
We found that R7 peptides are extraordinarily effective at translocating across the cytoplasmic membrane of vascular cells. We hypothesized that R7 itself, as a source of NO synthase substrate, might have an effect on myointimal hyperplasia. To test this hypothesis, an established rabbit vein graft model of myointimal hyperplasia was used. Interposition of a vein graft in an arterial circulation causes hemodynamic injury to the endothelium and vascular smooth muscle of the graft. Within 24 hours of vascular injury, VSMCs express iNOS.17 30 31 Under these conditions, vascular NO synthesis can be enhanced by exogenous L-arginine mainly via the action of iNOS. We found that brief (5-minute) treatment with L-R7 significantly increased local NO production and markedly reduced intimal expansion. We used both vehicle and L-R5 treatment as controls. Treatment for the same brief duration with the pentamers of L-R5 (which does not translocate across the cell membrane in the rapid fashion of L-R7) was no different from treatment with vehicle. This finding suggests that the observed inhibitory effects were due to translocation of the heptamers of arginine and not simply to the availability of polyarginine, perhaps complexed to the cell membrane.
The increase in NO synthesis induced by L-R7 was predictably associated with an inhibition of myointimal hyperplasia. The L-R7 polymer induced a 73% reduction in I/M thickness of the vein grafts, which was significantly greater than the 47% reduction by the D-arginine polymer. Nevertheless, because D-arginine is not a substrate for NO synthase, its modest inhibitory effect on myointimal hyperplasia was surprising. The effect of D-R7 also might have been due to NO production after epimerization of D- to L-arginine. Alternatively, D-arginine may be nonenzymatically oxidized to D-citrulline and NO by a nonenzymatic reaction involving hydrogen peroxide.32 It is also possible that some D-arginine was converted to the L form before its metabolism by iNOS.33 34
Alternatively, there may be an NO-independent mechanism(s) of R7 action. One possible NO-independent effect of the arginine polymers might be mediated by a cationic interaction with nucleic acid. It is possible that after nuclear translocation, highly positively charged arginine polymers interact with RNA in the nucleus and may interfere with the translation required for VSMC proliferation. Many proteins that interact with ribosomal RNA have arginine-rich sequences.35 36 These proteins are largely found in the nucleus and appear to be involved in RNA processing and transcriptional control. Typically, these residues are methylated by specific protein methyl arginine transferases.37 It is possible that R7 competes for these protein methyl arginine transferases or otherwise interferes with protein-RNA interaction. The aggregate data indicate that arginine polymers may inhibit VSMC proliferation by NO-dependent and -independent mechanisms.
In summary, short polymers of arginine (R7) are efficient at penetrating the vessel wall and translocating across the cytoplasmic membrane of vascular cells even while coupled to another molecule. These peptides may be efficient research tools for intracellular delivery of therapeutic agents. Furthermore, these compounds may have intrinsic biological effects on the vessel wall that may be useful in preventing or treating vascular disease.
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Acknowledgments |
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This study was supported in part by a Grant-in-Aid from the American Heart Association and by funding from CellGate, Inc. Dr Uemura is a recipient of a Fukuda Memorial Foundation Research Grant (Tokyo, Japan). Dr Cooke is an Established Investigator of the American Heart Association.
Received March 10, 2000; revision received June 22, 2000; accepted June 22, 2000.
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