(Circulation. 1999;99:111-120.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Age-Dependent Impairment of Angiogenesis
Presented in preliminary form as finalist presentation for the Melvin Marcus Young Investigator Award at the 70th Scientific Sessions of the American Heart Association, November 9–12, 1997, and published in abstract form (Circulation. 1997;96:I-J).
From the Departments of Medicine (Cardiology) and Biomedical Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass.
Correspondence to Jeffrey M. Isner, MD, St. Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail jisner{at}opal.tufts.edu
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Abstract |
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Background—The effect of aging on angiogenesis in ischemic vascular disease has not been studied. Accordingly, we investigated the hypothesis that angiogenesis is impaired as a function of age.
Methods and Results—Forty days after the resection of 1 femoral artery, collateral vessel development was significantly impaired in old (aged 4 to 5 years; n=7) versus young (aged 6 to 8 months; n=6) New Zealand White (NZW) rabbits on the basis of reduced hindlimb perfusion (ischemic: normal blood pressure ratio=0.58±0.05 versus 0.77±0.06; P<0.005), reduced number of angiographically visible vessels (angiographic score=0.48±0.05 versus 0.70±0.05; P<0.01), and lower capillary density in the ischemic limb (130.3±5.8/mm2 versus 171.4±9.5/mm2; P<0.001). Angiogenesis was also impaired in old (aged 2 years) versus young (aged 12 weeks) mice as shown by reduced hindlimb perfusion (measured by laser Doppler imaging) and lower capillary density (353.0±14.3/mm2 versus 713.3±63.4/mm2; P<0.01). Impaired angiogenesis in old animals was the result of impaired endothelial function (lower basal NO release and decreased vasodilation in response to acetylcholine) and a lower expression of vascular endothelial growth factor (VEGF) in ischemic tissues (by Northern blot, Western blot, and immunohistochemistry). When recombinant VEGF protein was administered to young and old rabbits, both groups exhibited a significant and similar increase in blood pressure ratio, angiographic score, and capillary density.
Conclusions—Angiogenesis responsible for collateral development in limb ischemia is impaired with aging; responsible mechanisms include age-related endothelial dysfunction and reduced VEGF expression. Advanced age, however, does not preclude augmentation of collateral vessel development in response to exogenous angiogenic cytokines.
Key Words: growth substances • endothelium • aging • angiogenesis
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Introduction |
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Advanced age is a major risk factor for coronary and peripheral artery disease.1 When vascular obstructions in either circulatory system are so extensive that direct revascularization techniques cannot be undertaken successfully, the severity of residual ischemia will depend in large part on the ability of the organism to spontaneously develop new collateral blood vessels. To the best of our knowledge, no study has previously evaluated the effect of aging on angiogenesis in ischemic vascular diseases. Moreover, the mechanisms by which aging could limit the formation of new blood vessels remain largely undefined.
Recent studies have demonstrated that angiogenesis, facilitated via administration of angiogenic growth factors as in recombinant protein therapy2 3 4 5 6 7 or gene transfer,8 9 10 may be augmented in animal models of myocardial and limb ischemia. The impact of aging in these experimental models, however, was not tested. This issue may have important implications for the utility of such therapeutic strategies in older patients who indeed represent the population subset most likely to benefit from such therapies. Accordingly, the present study was designed to investigate the hypothesis that angiogenesis is impaired as a function of age and, if confirmed, to identify potentially contributory mechanisms.
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Methods |
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Animal Models
All protocols were approved by St. Elizabeth's Institutional Animal Care and Use Committee. The development of angiogenesis in response to regional ischemia was investigated in 2 animal models.
Rabbit Ischemic Hindlimb Model
The first animal model involved young (aged 6 to 8 months)
versus old (aged 4 to 5 years) New Zealand White (NZW) rabbits in which
operative intervention was performed to establish unilateral hindlimb
ischemia. The maximum age for NZW rabbits has been previously
reported to be 7 years.11
Surgery
Male NZW rabbits (weight, 3.6 to 4 kg) were anesthetized
with a mixture of ketamine (50 mg/kg) and acepromazine (0.8
mg/kg) after premedication with xylazine (2 mg/kg). Young (n=6) and old
(n=7) rabbits underwent operative resection of 1 femoral artery as
previously described.2 3 9 In order that these
groups could also serve as a basis for comparison with animals
receiving exogenous cytokine therapy, all 13 rabbits received
saline with 0.1% rabbit serum albumin (Sigma Chemical Co)
administered via an intra-arterial route (vide infra) on
postoperative day 10.
Administration of Supplemental Angiogenic Cytokines
An additional 14 NZW rabbits (7 young and 7 old) received
recombinant human vascular endothelial growth factor
(VEGF) (500 µg of rhVEGF165)in the proximal
segment of the internal iliac artery of the ischemic limb as
described previously2 on postoperative day
10.
Hindlimb Perfusion Pressure
Blood pressure (BP) was measured as previously
described.2 The ratio of ischemic to
normal hindlimb BP (BPR) was defined for each rabbit as the ratio of
systolic pressure measured in the ischemic limb to
systolic pressure measured in the normal limb.
Angiography
Selective angiography of the ischemic hindlimb was
performed on days 10 and 40 after surgery as previously
described.2 3 9 The luminal diameter of the
internal iliac artery was measured with a validated automated
edge-detection system (CatView, Imagecom). To quantitatively assess
collateral vessel development, we used an acetate overlay with an
imprinted grid composed of 2.5-mm-diameter circles arranged in rows
spaced 5 mm apart to yield an angioscore as previously
described.2
Capillary Density
Tissue specimens obtained as transverse sections from the
adductor and semimembranous muscle groups of both limbs of each rabbit
at the time of death (day 40) were embedded in OCT compound (Miles) and
snap-frozen in liquid nitrogen. Tissue sections were stained for
alkaline phosphatase by an indoxyl-tetrazolium method to detect
capillary endothelial cells and were then
counterstained with eosin.
Measurement of Nitrite Production From Rabbit Aortas Ex
Vivo
Aortas were rapidly isolated after extensive washing with
saline, with care being taken to preserve the
endothelium intact. The retrieved aortic segments were
immersed in oxygenated Krebs buffer. After 5 minutes of
equilibration, Krebs buffer (10 mL) was replaced and incubated for an
additional 10 minutes. Nitrite concentration was measured by Griess
reaction as described previously12 and was
expressed as picomoles per square millimeter of
endothelial surface area.
Murine Ischemic Hindlimb Model
Surgery
Unilateral hindlimb ischemia was created in C57BL/6
female mice13 14 that were 12 weeks (young) or 2
years (old) of age. The animals were anesthetized with
pentobarbital (160 mg/kg IP), after which an incision was performed in
the skin overlying the middle portion of the left hindlimb. After
ligation of the proximal end of the femoral artery, the distal portion
of the saphenous artery was ligated, and the artery and all side
branches were dissected free and excised. The skin was closed with a
surgical stapler.
Monitoring of Hindlimb Blood Flow
Hindlimb perfusion was measured with a laser Doppler
perfusion imager (LDPI) system (Lisca Inc). After
anesthesia, consecutive measurements were obtained after
scanning of the same region of interest (leg and foot) with the LDPI.
The perfusion signal was split into 6 different intervals, each
displayed in a separate color. Low or no perfusion was displayed in
dark blue, whereas the highest perfusion interval was displayed in red.
The stored perfusion values behind the color-coded pixels were then
available for analysis. To account for variables such as
ambient light and temperature, the results are expressed as the ratio
of perfusion in the left (ischemic) versus right (normal)
limb.
Tissue Preparation
The mice were killed at predetermined arbitrary time points
after surgery with an overdose of sodium pentobarbital. For
immunohistochemistry, whole ischemic and nonischemic
limbs were immediately fixed in methanol overnight. After bones had
been carefully removed, 3-µm-thick tissue sections were cut and
paraffin-embedded. For total protein and RNA extraction, isolated
tissue samples were rinsed in PBS to remove excess blood, snap-frozen
in liquid nitrogen, and stored at -80°C until use.
Immunohistochemistry
Histological sections (5 µm thick)
prepared from paraffin-embedded tissue samples of the lower limbs were
used for immunohistochemical analysis. Identification of
endothelial cells was performed by immunohistochemical
staining for platelet endothelial cell adhesion
molecule-1 (PECAM-1 or CD31) with a rat monoclonal antibody directed
against mouse CD31 (Pharmingen). Identification and localization of T
lymphocytes in tissues were performed by immunohistochemical staining
for CD3 (a pan T-cell surface marker) with a polyclonal anti-human CD3
antibody (Sigma). Immunohistochemical localization of VEGF was
performed with a rabbit polyclonal antibody directed against human VEGF
amino-terminal peptides 1 through 20 (Santa Cruz Biotechnology) that
cross-reacts with murine VEGF. Immunoperoxidase staining was performed
as previously described.14 15
Analysis of Capillary Density
Capillaries, identified by positive staining for CD31 and
appropriate morphology, were counted by a single observer blinded to
the treatment regimen under a 20x objective and a 5x lens to
determine the capillary density (mean number of capillaries per square
millimeter).2 A total of 20 different fields from
the 2 muscles were randomly selected, and the number of capillaries was
counted for each field.
Quantification of T Lymphocytes in Ischemic Tissues
The number of CD3-positive cells was counted by a single
observer under a 20x objective and a 5x lens. A minimum of 10
different fields from the ischemic tissues of mice were
randomly selected, and the number of T lymphocytes was counted for each
field. The results are expressed as the average of T lymphocytes per
high-power field.
Northern Blot Analysis of VEGF mRNA Expression
Total tissue RNA was isolated from ischemic hindlimb
muscles of mice by phenol/chloroform
extraction.16 Twenty micrograms of RNA per lane
was separated by electrophoresis on 1% agarose gel containing
formaldehyde and transferred to a nylon membrane (Hybond-N, Amersham)
by blotting. The membrane was hybridized with
32P-labeled probe specific for VEGF, a 675-bp
EcoRI/BglII fragment of plasmid
pSVI.VEGF.21.17 Hybridization was performed as
previously described.16
Western Blot Analysis of VEGF Protein Expression
Whole-cell protein extracts were obtained after
homogenization of ischemic and control
muscles of both young and old animals. A total of 200 µg of protein
per sample was separated on a 12% polyacrylamide gel and
electroblotted on nitrocellulose membranes.18 The
membrane was blocked with 10% nonfat dry milk in 0.2% Tween PBS
(T-PBS) and then probed with 1:250 of rabbit polyclonal anti-human VEGF
antibody (Sigma) for 3 hours at room temperature. After incubation with
primary antibody, the blot was washed three times in T-PBS and was then
incubated for 1 hour with 1:4000 of anti-rabbit horseradish peroxidase
IgG (Santa Cruz Biotechnology). The blot was then washed in T-PBS, and
antigen-antibody complexes were visualized after incubation for 1
minute with enhanced luminescence reagent (Amersham) at room
temperature, followed by exposure to Kodak XAR-5 film.
T-Cell Fluorescent-Activated Cell Sorting
Analysis
Fluorescent-activated cell sorting (FACS)
analysis was performed on a FACScan (Becton-Dickinson); >5000
cells were analyzed per sample. Mouse blood was obtained by
intracardiac puncture and placed in EDTA, and buffy coats were
separated over Histopaque (Sigma). The cells were double-stained with
phycoerythrin anti-mouse CD3 (Pharmingen). The cells were washed
again and fixed with paraformaldehyde (1%). The
absolute number of T-cells was calculated from the percentage of CD3
positive cells of each subset multiplied by the total number of
leukocytes.
VEGF Promoter Activity
Vascular smooth muscle cells (VSMCs) were isolated from the
aortas of young and old rabbits, seeded into 6-well plates, and
maintained in DMEM supplemented with 10% FBS. The next day, cells
(60% to 80% confluence) were transiently transfected with 10 µg of
a reporter construct containing the luciferase gene under the
transcriptional control of the VEGF promoter and 30 µg of
Lipofectamine reagent (GIBCO Laboratories). To correct for
differences in transfection efficiency, luciferase activity was
normalized relative to the level of alkaline phosphatase activity
produced from cotransfected pSVAPAP plasmid (0.5 µg), which contains
the reporter gene under the control of the simian virus 40
enhancer-promoter. The cells were incubated with the transfection
mixture for 3 hours and then were fed with low-serum (0.25% FBS) or
high-serum (10% FBS) DMEM. After 24 hours, luciferase and alkaline
phosphatase activities were measured in old and young VSMCs. Results
are expressed as the ratio of luciferase to alkaline phosphatase
activities.
Statistical Analysis
All results are expressed as mean±SEM. Statistical significance
was evaluated by ANOVA or 2-tailed unpaired Student's t
test for comparisons between the mean of 2 groups. A value of
P<0.05 was interpreted to denote statistical
significance.
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Results |
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Postsurgical Follow-Up
The consequences of hindlimb ischemia were more profound in the old animals. This was especially apparent in the old mice; severe necrosis of the distal part of the ischemic limb was noted in 5 of the 6 mice studied (Figure 1
). In comparison, none of the 9 young
mice studied developed limb necrosis.
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Analysis of Native Angiogenesis
As shown in Figure 2, calf BPR was
similar in both old (n=7) and young (n=6) rabbits at day 10
postoperatively. By day 40, however, BPR improvement was significantly
greater in young than in old rabbits (0.77±0.06 versus 0.58±0.05;
P=0.02). Both young and old rabbits had a significant and
similar increase in BPR when treated with
rhVEGF165 protein. In young treated rabbits, BPR
increased from 0.77±0.06 to 0.92±0.04 (P=0.03). In old
rabbits, BPR increased from 0.58±0.05 to 0.75±0.05
(P=0.03). The ultimate level of BPR achieved by the old
rabbits treated with rhVEGF165 protein was
significantly lower than that of the young treated rabbits (0.75±0.05
versus 0.92±0.04; P=0.02).
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Perfusion of the ischemic hindlimb in mice assessed by laser
Doppler measurement (Figure 3) was
also reduced in old (n=6) compared with young (n=9) mice. At day 7
after surgery, the Doppler flow ratio was significantly reduced in
old mice (0.1±0.02 versus 0.26±0.04; P=0.014), and this
difference was exacerbated at day 28 after surgery (0.23±0.03 versus
0.65±0.06; P=0.0005). This severe impairment of blood flow
caused necrosis and autoamputation of the ischemic foot in old
mice.
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The number of angiographically visible collateral vessels (angiographic
score) was markedly reduced in old versus young rabbits (Figure 4). At day 10, angiographic scores were
similarly low for both groups (P=NS). At day 40, however,
angiography disclosed significantly fewer collateral vessels in the
medial thigh area of old compared with young NZW rabbits
(angioscore=0.48±0.05 versus 0.70±0.05; P=0.008).
Treatment with rhVEGF165 protein resulted in a
significant and similar increase in the number of angiographically
visible collaterals in both young and old rabbits (Figure 4F). The
angioscore for young animals treated with
rhVEGF165 (0.91±0.08) was significantly higher
than the corresponding value (0.70±0.05) obtained in untreated animals
(P=0.03). In old treated rabbits, the angioscore
(0.69±0.04) was also significantly higher than that recorded for
the untreated group (0.48±0.05; P=0.005). The ultimate
magnitude of angiographically visible collaterals observed in the old
treated rabbits, however, remained inferior to that of the
young treated rabbits (0.69±0.04 versus 0.91±0.08;
P=0.014)
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Tissue sections from the medial thigh muscles of rabbits were examined
histologically at day 40 as described above. As shown
in Figure 5A and 5B, capillary density
was significantly lower in old NZW rabbits
(130.3±5.8/mm2) than in young rabbits
(171.4±9.5/mm2; P<0.001). Likewise,
capillary density as assessed by CD31 immunostaining
was also reduced in old versus young mice
(353.0±14.3/mm2 versus
713.3±63.4/mm2; P<0.01) at 28 days
after surgery (Figure 5E, 5F, and 5H). Supplemental
rhVEGF165 induced a significant
(P<0.001) increase in capillary density in both old and
young rabbits (Figure 5C, 5D, and 5G) (191.4±7.8 and
282.7±5.0/mm2, respectively, compared with
130.3±5.8/mm2 and
171.4±9.5/mm2 in the untreated groups). There
was no statistically significant difference (P=NS) between
old and young rabbits in the magnitude of improvement observed for
these end points after rhVEGF165 treatment.
However, the ultimate level of capillary density achieved in the
ischemic hindlimbs of VEGF treated animals was still lower in
the old rabbits than in the young rabbits (P<0.001).
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Vasomotor Reactivity
Because endothelial cells constitute the principal
cellular element responsible for
neovascularization,19 we considered that
dysfunctional endothelial cells could represent
a putative basis for age-dependent angiogenesis. To assess the
integrity of endothelial function in vivo, the
magnitude of vasorelaxation induced by the
endothelium-dependent agonist acetylcholine was
determined by angiography in untreated rabbits. As shown in Figure 6A, vasorelaxation induced by
acetylcholine was significantly (P<0.05) reduced in old
versus young NZW rabbits (3.9±01.3% versus 8.8±1.8%). That this was
not due simply to a generic reduction in vasomotor responsivity was
demonstrated by equivalent vasorelaxation in response to nitroprusside
for both groups of rabbits (4.1±3.8% versus 3.1±2.1%;
P=NS).
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Measurement of NO
To further characterize the extent of endothelial
dysfunction in old versus young rabbits, we measured NO
production from freshly isolated aortic rings. In aortic rings
from old rabbits, NO production was significantly reduced
compared with that in young rabbits (Figure 6B
), with nitrite values of
31.6±3.6 versus 158.4±54.8 pmol/mm2,
respectively (P<0.03).
VEGF mRNA Expression
Expression of VEGF mRNA in ischemic tissues was markedly
reduced in old versus young mice. The difference in mRNA levels between
the 2 groups was especially apparent at day 7 and day 14 after
development of limb ischemia, as shown on the Northern blot in
Figure 7.
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VEGF Protein Expression in Tissues From Ischemic and
Control Limbs
Endogenous expression of VEGF protein was determined
for old and young animals by Western blot analysis of protein
extracts obtained from muscles of both the ischemic and normal
limbs harvested at different time points after surgery. Figure 8A shows the expression of VEGF protein
in young and old rabbits in ischemic muscles harvested 7 days
after hindlimb surgery. The level of VEGF protein was significantly
reduced in old versus young rabbits. Similar results were obtained in
old and young mice: the upper panel of Figure 8B shows that basal
expression of VEGF is low in the nonischemic limbs of
unoperated mice. There were no significant differences in basal
expression of VEGF in young versus old mice. The lower panel of Figure 8B shows the time course of VEGF expression in young and old
mice after operative induction of hindlimb ischemia.
VEGF was upregulated as early as day 3 after surgery, reached a maximum
by day 7, and decreased thereafter. The level of VEGF protein was
significantly reduced in old versus young mice for all time points
studied. Immunostaining confirmed the results of the
Western blot by showing a lower level of VEGF expression in the tissues
retrieved from old versus young mice at day 7 after surgery (Figure 8C). Tissue immunohistochemistry further established that the cell
types responsible for VEGF expression included skeletal myocytes and T
lymphocytes infiltrating the ischemic tissues (Figure 9).
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T Cells in Ischemic Tissues
Immunostaining for CD3 revealed a lower number of
infiltrating T cells (3.3±0.2 versus 11.7±1.8 per high-power field,
P<0.05) in old mice than in young mice (Figure 9). This
difference in T-cell infiltrate could not be attributed simply to a
reduction in circulating peripheral blood T cells. Although
the total number of white blood cells was lower in old animals, FACS
analysis performed in 10 unoperated mice (5 young and 5 old)
established that the absolute number (as opposed to the percentage) of
peripheral blood T cells was similar in both groups (Figure 9D).
VEGF Promoter Activity
Using VSMCs isolated from aortas of young or old rabbits, we
studied the expression of the VEGF promoter in low- or high-serum
conditions. The cells were transfected with a plasmid that contains the
firefly luciferase gene under the control of the VEGF promoter. As seen
in Figure 10, although the VEGF
promoter was induced by serum in both young and old VSMCs, the level of
promoter activity was dramatically reduced in old cells, especially in
high-serum conditions. This result implies that aging impairs VEGF
expression at the transcriptional level.
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Discussion |
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The results of the present experiments establish that angiogenesis is impaired as a function of age. The reduced capability for collateral vessel development in response to ischemia was confirmed in 2 different animal models. The ultimate hindlimb BPR achieved at 40 days after surgery was significantly less in old than in young NZW rabbits. In old mice, perfusion of the ischemic hindlimb, reflected by the Doppler flow ratio, was significantly reduced compared with young mice; this difference was apparent as soon as 7 days after surgery and persisted throughout the duration (28 days) of the study. Likewise, the number of blood vessels that were angiographically visible in rabbits and the number of capillaries per unit area identified histologically in mice and rabbits were both significantly reduced in old versus young animals. The latter finding is consistent with the observation that myocardial angiogenesis related to left ventricular hypertrophy is attenuated in an age-dependent manner.20
The mechanisms by which aging can affect angiogenesis are potentially diverse. Angiogenesis is a complex process that includes activation, migration, and proliferation of endothelial cells.19 Recent studies21 22 23 24 have indicated that the integrity of endothelial cell function may be compromised as a function of advanced age. We confirmed that endothelial function was abnormal in old versus young rabbits by documenting impaired vasodilation in response to the endothelium-dependent vasodilator acetylcholine in vivo and reduced release of NO from isolated blood vessels studied ex vivo. Previous in vitro studies25 26 have also suggested that aged endothelial cells show impaired proliferation and migration in response to cytokines such as platelet-derived growth factor and fibroblast growth factor. Taken together, these observations support the notion that age-dependent endothelial dysfunction contributes to impaired angiogenesis in the setting of tissue ischemia.
Growth factors, particularly endothelial cell mitogens, represent a second essential element in the promotion and regulation of angiogenesis. Numerous reports suggest that VEGF, an endothelial cell–specific mitogen, is a critical growth factor in therapeutic2 3 6 7 9 10 and pathological27 28 29 angiogenesis. In the present study, we demonstrated that the magnitude of VEGF expression in tissues harvested from ischemic limbs of old mice and rabbits was reduced compared with that observed in young animals.
The observed reduction in VEGF expression appears to be at least bifactorial. First, immunostaining of tissue specimens harvested from the murine hindlimbs disclosed less VEGF protein expression in skeletal myocytes from old versus young mice. Moreover, the finding that VEGF promoter activity was reduced in old versus young VSMCs suggests that the reduction in VEGF expression observed in old animals is due at least in part to a defect in transcriptional regulation. Second, T lymphocytes, shown immunohistochemically to constitute a source of VEGF protein in young mice, were markedly reduced in tissue sections retrieved from old mice and stained with antibodies to CD3. T cells have previously been shown to constitute a potentially important source of VEGF that contributes to the growth of malignant neoplasms.30 More recently, studies performed in our own laboratory have shown that the development of hindlimb ischemia in nude mice is quickly followed by limb necrosis and autoamputation (T. Couffinhal, MD, unpublished data, 1996). These findings thus reinforce the potential contribution of T cells to VEGF expression in the setting of tissue ischemia and are consistent with the interpretation that the lower level of T cells detected in the ischemic hindlimbs of old mice may be responsible, at least in part, for the local reduction in VEGF expression. Although conflicting results have been reported in human studies regarding the effects of age on the level of CD4+ cells,31 the notion that T-cell immunity is compromised as a function of age is well documented. T-cell proliferation in vitro and in vivo declines with age in both mice and humans, and there is a shift away from naive CD4+ cells toward a relative increase in memory subsets.32
The fact that we observed fewer T cells in the ischemic tissues of older mice despite similar peripheral T-cell counts in old and young mice is consistent with the notion of an age-dependent defect in transendothelial migration of T lymphocytes33 into the target ischemic tissues. Although the precise mechanisms responsible for T-cell migration remain enigmatic, recent reports suggest that a combination of signals is required to trigger the migratory T-cell phenotype34 and that CD4+ activated T cells are more likely to transmigrate than CD4- cells.35
These observations suggest that age-related reduction in activated T cells migrating into the tissues of the ischemic limbs may obviate a source of VEGF that is potentially important to upregulate expression of this angiogenic growth factor in the setting of limb ischemia. This finding may in fact represent a conceptual link to studies performed in mice injected with tumor cells in which old animals were shown to have a slower rate of tumor growth than younger animals; such altered tumor growth was associated with a reduced capacity to vascularize the tumors.36 37 Indeed, others38 have suggested that age-dependent reduction in tumor vascularization may result from a lower level of "lymphocyte-induced angiogenesis factor."
The favorable response to VEGF replacement therapy described above strongly implicates VEGF as the pivotal cytokine deficiency responsible for impaired angiogenesis. This interpretation is consistent with the fact that absence of a single VEGF allele in the developing embryo is sufficient to prohibit vascular development and with the finding that VEGF appears to lie downstream of several, if not all, other angiogenic cytokines.17 39 40 41 Involvement of other angiogenic growth factors cannot be excluded on the basis of the data given in the present study. Similarly, the present findings do not exclude the possibility that age-impaired angiogenesis is due in part to upregulated expression of a natural endogenous inhibitor of angiogenesis,29 although experiments recently performed in our own laboratory have failed to provide evidence that impaired angiogenesis is associated with the candidate inhibitor thrombospondin (C. Kalka, MD, unpublished data, 1997).
The magnitude of improvement in end points used to assess limb perfusion after rhVEGF165 replacement therapy was similar for old and young rabbits. This finding suggests that the expression and function of VEGF receptors are preserved in old animals. Indeed, we have found that expression of the principal VEGF receptor KDR, as assessed by Western blotting, was not reduced in normal hindlimbs of old mice compared with young mice (data not shown).
However, the ultimate level of recovery achieved in old animals after rhVEGF165 therapy was still inferior to that observed in young rabbits after identical treatment. This finding suggests that persistent endothelial dysfunction may represent the rate-limiting factor that affects angiogenesis. Additional studies are therefore required to characterize the full complement of responsible mechanisms that might allow optimization of strategies designed to address critical limb ischemia in the expanding population of elderly individuals.
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Acknowledgments |
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This study was supported in part by an Academic Award in Vascular Medicine (HL-02824) and grants from the National Institutes of Health (HL-53354 and HL-57516). Dr Rivard is supported by a grant from the Heart and Stroke Foundation of Canada.
Received May 5, 1998; revision received August 19, 1998; accepted September 2, 1998.
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E. G. Lakatta and S. Schulman Age-associated cardiovascular changes are the substrate for poor prognosis with myocardial infarction J. Am. Coll. Cardiol., July 7, 2004; 44(1): 35 - 37. [Full Text] [PDF]
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J.-H. Choi, K. L. Kim, W. Huh, B. Kim, J. Byun, W. Suh, J. Sung, E.-S. Jeon, H.-Y. Oh, and D.-K. Kim Decreased Number and Impaired Angiogenic Function of Endothelial Progenitor Cells in Patients With Chronic Renal Failure Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1246 - 1252. [Abstract] [Full Text] [PDF]
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P. D. Lambiase, R. J. Edwards, P. Anthopoulos, S. Rahman, Y. G. Meng, C. A. Bucknall, S. R. Redwood, J. D. Pearson, and M. S. Marber Circulating Humoral Factors and Endothelial Progenitor Cells in Patients With Differing Coronary Collateral Support Circulation, June 22, 2004; 109(24): 2986 - 2992. [Abstract] [Full Text] [PDF]
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D. W. Losordo and S. Dimmeler Therapeutic Angiogenesis and Vasculogenesis for Ischemic Disease: Part II: Cell-Based Therapies Circulation, June 8, 2004; 109(22): 2692 - 2697. [Full Text] [PDF]
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L. Lawrenson, J. Hoff, and R. S. Richardson Aging attenuates vascular and metabolic plasticity but does not limit improvement in muscle VO2 max Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1565 - H1572. [Abstract] [Full Text] [PDF]
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A. L. Serrano and V. Andres Telomeres and Cardiovascular Disease: Does Size Matter? Circ. Res., March 19, 2004; 94(5): 575 - 584. [Abstract] [Full Text] [PDF]
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R. J. Scheubel, H. Zorn, R.-E. Silber, O. Kuss, H. Morawietz, J. Holtz, and A. Simm Age-dependent depression in circulating endothelial progenitor cells inpatients undergoing coronary artery bypass grafting J. Am. Coll. Cardiol., December 17, 2003; 42(12): 2073 - 2080. [Abstract] [Full Text] [PDF]
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S. Dimmeler and M. Vasa-Nicotera Aging of progenitor cells: limitation for regenerative capacity? J. Am. Coll. Cardiol., December 17, 2003; 42(12): 2081 - 2082. [Full Text] [PDF]
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A. Germani, A. Di Carlo, A. Mangoni, S. Straino, C. Giacinti, P. Turrini, P. Biglioli, and M. C. Capogrossi Vascular Endothelial Growth Factor Modulates Skeletal Myoblast Function Am. J. Pathol., October 1, 2003; 163(4): 1417 - 1428. [Abstract] [Full Text] [PDF]
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T. Koike, R. B. Vernon, M. D. Gooden, E. Sadoun, and M. J. Reed Inhibited Angiogenesis in Aging: A Role for TIMP-2 J. Gerontol. A Biol. Sci. Med. Sci., September 1, 2003; 58(9): B798 - 805. [Abstract] [Full Text] [PDF]
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C. Patterson The Ponzo Effect: Endothelial Progenitor Cells Appear on the Horizon Circulation, June 24, 2003; 107(24): 2995 - 2997. [Full Text] [PDF]
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H. Masuda and T. Asahara Post-natal endothelial progenitor cells for neovascularization in tissue regeneration Cardiovasc Res, May 1, 2003; 58(2): 390 - 398. [Abstract] [Full Text] [PDF]
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N. van Royen, I. Hoefer, M. Bottinger, J. Hua, S. Grundmann, M. Voskuil, C. Bode, W. Schaper, I. Buschmann, and J.J. Piek Local Monocyte Chemoattractant Protein-1 Therapy Increases Collateral Artery Formation in Apolipoprotein E-Deficient Mice but Induces Systemic Monocytic CD11b Expression, Neointimal Formation, and Plaque Progression Circ. Res., February 7, 2003; 92(2): 218 - 225. [Abstract] [Full Text] [PDF]
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E. G. Lakatta Arterial and Cardiac Aging: Major Shareholders in Cardiovascular Disease Enterprises: Part III: Cellular and Molecular Clues to Heart and Arterial Aging Circulation, January 28, 2003; 107(3): 490 - 497. [Full Text] [PDF]
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J. M. Edelberg Auto Repair on the Aging Stem Cell Superhighway Sci. Aging Knowl. Environ., September 4, 2002; 2002(35): pe13 - 13. [Abstract] [Full Text]
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Z. S. Katusic Therapeutic Angiogenesis: New Indication for Endothelial NO Synthase Gene Transfer Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1254 - 1255. [Full Text] [PDF]
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J. L. Tuttle, T. L. Hahn, B. M. Sanders, F. A. Witzmann, S. J. Miller, M. C. Dalsing, and J. L. Unthank Impaired collateral development in mature rats Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H146 - H155. [Abstract] [Full Text] [PDF]
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T. Krabatsch, R. Petzina, H. Hausmann, A. Koster, and R. Hetzer Factors influencing results and outcome after transmyocardial laser revascularization Ann. Thorac. Surg., June 1, 2002; 73(6): 1888 - 1892. [Abstract] [Full Text] [PDF]
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J. M. Edelberg, L. Tang, K. Hattori, D. Lyden, and S. Rafii Young Adult Bone Marrow-Derived Endothelial Precursor Cells Restore Aging-Impaired Cardiac Angiogenic Function Circ. Res., May 31, 2002; 90 (10): e89 - e93. [Abstract] [Full Text] [PDF]
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S. Marchetti, C. Gimond, K. Iljin, C. Bourcier, K. Alitalo, J. Pouyssegur, and G. Pages Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo J. Cell Sci., May 15, 2002; 115(10): 2075 - 2085. [Abstract] [Full Text] [PDF]
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J. M. Edelberg, J. T. Jacobson, D. S. Gidseg, L. Tang, and D. J. Christini Enhanced myocyte-based biosensing of the blood-borne signals regulating chronotropy J Appl Physiol, February 1, 2002; 92(2): 581 - 585. [Abstract] [Full Text] [PDF]
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T. Pohl, C. Seiler, M. Billinger, E. Herren, K. Wustmann, H. Mehta, S. Windecker, F. R. Eberli, and B. Meier Frequency distribution of collateral flow and factors influencing collateral channel development: Functional collateral channel measurement in 450 patients with coronary artery disease J. Am. Coll. Cardiol., December 1, 2001; 38(7): 1872 - 1878. [Abstract] [Full Text] [PDF]
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K. Przyklenk, G. Li, and P. Whittaker No loss in the in vivo efficacy of ischemic preconditioning in middle-aged and old rabbits J. Am. Coll. Cardiol., November 15, 2001; 38(6): 1741 - 1747. [Abstract] [Full Text] [PDF]
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P. Abete, N. Ferrara, F. Cacciatore, E. Sagnelli, M. Manzi, V. Carnovale, C. Calabrese, D. de Santis, G. Testa, G. Longobardi, et al. High level of physical activity preserves the cardioprotective effect of preinfarction angina in elderly patients J. Am. Coll. Cardiol., November 1, 2001; 38(5): 1357 - 1365. [Abstract] [Full Text] [PDF]
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S. Takeshita, H. Tomiyama, N. Yokoyama, Y. Kawamura, T. Furukawa, Y. Ishigai, T. Shibano, T. Isshiki, and T. Sato Angiotensin-converting enzyme inhibition improves defective angiogenesis in the ischemic limb of spontaneously hypertensive rats Cardiovasc Res, November 1, 2001; 52(2): 314 - 320. [Abstract] [Full Text] [PDF]
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A. S. Khan, C. D. Lynch, D. C. Sane, M. C. Willingham, and W. E. Sonntag Growth Hormone Increases Regional Coronary Blood Flow and Capillary Density in Aged Rats J. Gerontol. A Biol. Sci. Med. Sci., August 1, 2001; 56(8): B364 - 371. [Abstract] [Full Text] [PDF]
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C. Emanueli, M. B. Salis, T. Stacca, L. Gaspa, J. Chao, L. Chao, A. Piana, and P. Madeddu Rescue of Impaired Angiogenesis in Spontaneously Hypertensive Rats by Intramuscular Human Tissue Kallikrein Gene Transfer Hypertension, July 1, 2001; 38(1): 136 - 141. [Abstract] [Full Text] [PDF]
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M. Simons Therapeutic coronary angiogenesis: a fronte praecipitium a tergo lupi? Am J Physiol Heart Circ Physiol, May 1, 2001; 280(5): H1923 - H1927. [Full Text] [PDF]
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J. Waltenberger Impaired collateral vessel development in diabetes: potential cellular mechanisms and therapeutic implications Cardiovasc Res, February 16, 2001; 49(3): 554 - 560. [Abstract] [Full Text] [PDF]
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E. VASILE, Y. TOMITA, L. F. BROWN, O. KOCHER, and H. F. DVORAK Differential expression of thymosin {beta}-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis FASEB J, February 1, 2001; 15(2): 458 - 466. [Abstract] [Full Text] [PDF]
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