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¶
From the Department of Molecular Medicine* andthe National Bio-NMR Facility, A. I. Virtanen Institute, and the Departments ofSurgery andMedicine,¶ Kuopio University, and the Gene TherapyUnit, Kuopio University Hospital,Kuopio, Finland
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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was associated with VEGF and VEGFR-2
expression both in acute and chronic ischemia but not in regeneration.
Hindlimb ischemia was induced in 20 New Zealand White rabbits by
excising the femoral artery. Magnetic resonance imaging and
histological sections revealed extensive ischemic damage in the thigh
and leg muscles of ischemic rabbit hindlimbs with VEGF expression
similar to acute human lower limb ischemia. After 1 and 3 weeks of
ischemia VEGF expression was restricted to regenerating myotubes and by
6 weeks regeneration and expression of VEGF was diminished. VEGFR-2
expression was co-localized with VEGF expression in regenerating
myotubes. Macrophages and an increased number of capillaries were
associated with areas of ischemic muscle expressing VEGF and VEGFR-2.
In conclusion, two patterns of VEGF and VEGFR-2 expression in
human and rabbit ischemic skeletal muscle are demonstrated. In acute
skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in
the affected muscle. In chronic skeletal muscle ischemia and in
skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression
are restricted to atrophic and regenerating muscle cells suggesting the
operation of an autocrine pathway that may promote survival and
regeneration of myocytes.
VEGF is expressed as at least five isoforms consisting of polypeptides
with 121-, 145-, 165-, 189-, or 206-amino-acid residues, differing in
their extracellular matrix-binding properties.3
VEGF
expression is induced by hypoxia, hypoglycemia, inflammation, tissue
repair, and malignancy, but many signal transduction pathways that
regulate VEGF expression remain unknown. It is known
that hypoxia and hypoglycemia induce VEGF expression by
increasing its transcription and stabilizing VEGF
mRNA.15
Hypoxia-inducible factor-1 (HIF-1) is the
main regulator of VEGF expression under different oxygen
concentrations.16
Growth factors such as platelet-derived
growth factor and fibroblast growth factor-2 also stimulate VEGF
synthesis synergistically with hypoxia.17,18
Previously, VEGF has been thought to be an endothelial cell-specific mitogen, but recent reports show that it may have multiple roles in vivo. For example, it enhances chemotaxis and migration of vascular smooth muscle cells, coordinates longitudinal bone growth and endochondral bone formation, is critical for the development of retinal cells and a survival factor for renal tubular epithelial cells, and it has a direct survival effect on ischemic neuronal cells in vitro.19-23 An important feature of VEGF is its chemotactic effect on circulating endothelial precursor cells and monocytes, which are inducers of vascular growth.24,25 Thus, apart from angiogenesis VEGF may induce other effects promoting the recovery of ischemic tissues.14
Although several clinical trials have already been started with VEGF protein or gene therapy,26 little information is available about the expression patterns of VEGF and VEGFR-2 in ischemic human skeletal muscle and whether animal models bear any resemblance to the human situation. Here we demonstrate the expression of VEGF, and for the first time, the expression of VEGFR-2 in skeletal myocytes of critically ischemic human lower limbs and in the rabbit model of hindlimb ischemia. It is shown that in acute ischemia widespread VEGF and VEGFR-2 expression is present whereas in chronic ischemia only atrophic and regenerating muscle cells express VEGF and VEGFR-2.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Ischemic human skeletal muscle samples were obtained from
eight patients after lower limb amputation because of critical
ischemia. Characteristics of the patients and muscle samples are shown
in Table 1
. Six patients suffered from
chronic critical limb ischemia, defined as ischemia resulting in either
long-lasting rest-pain (>2 to 4 weeks) and/or tissue loss. Two
patients had acute lower limb ischemia because of either an embolism or
a major thrombosis with occlusive atherosclerosis. Samples were
collected from two different regions of each amputee: one sample was
collected from a region that represented macroscopically the healthiest
part of the amputee, and the other one was chosen from a region that
showed more profound ischemia on the basis of the color and integrity
of muscle. Studies of human tissues were approved by the Ethical
Committee of Kuopio University Hospital.
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Twenty New Zealand White rabbits were anesthetized with fentanyl-fluanisone (0.2 ml/kg, Hypnorm; Janssen, Beerse, Belgium) and midazolam (1.5 mg/kg, Dormicum; Roche, Basel, Switzerland). Unilateral ischemia was induced by surgically removing the superficial femoral artery and ligating the deep femoral and lateral femoral circumflex arteries as previously described.27 All animals received prophylactic cefuroxim antibiotics (125 mg,Zinacef; GlaxoWellcome, Research Triangle Park, NC) before surgery. Bubrenorphine (Temgesic, 0.03 mg/kg, Schering-Plough, Kenilworth, NJ) was given as needed for postoperative pain. Rabbits were sacrificed 3 days and 1, 3, or 6 weeks after the operation (n = 5 in each group) and muscle samples were collected from tibialis anterior and soleus muscles in the leg and rectus and adductor muscles in the thigh for immunohistochemistry and in situ hybridization. Before the termination of the follow-up period selective internal iliac angiography was performed to visualize collateral artery growth. All animal experiments were approved by Experimental Animal Committee of Kuopio University.
Immunohistochemistry
Human and rabbit skeletal muscle samples were
immersion-fixed in 4% paraformaldehyde and 15% sucrose (pH 7.4) for 4
hours, rinsed in 15% sucrose (pH 7.4) overnight, and embedded with
paraffin.28
Six-µm-thick sections were prepared and
immunohistochemistry was done using the avidin-biotin-horseradish
peroxidase system (Vector Laboratories, Burlingame, CA). Capillaries
were immunostained with a monoclonal antibody (mAb) against human CD31
(dilution 1:50; DAKO, Glostrup, Denmark). Macrophages in rabbit
skeletal muscle were stained with a mAb against rabbit macrophages
(1:50, RAM-11; DAKO) and human macrophages with a mAb against human
CD-68 (1:100, DAKO). Skeletal myocytes were immunostained with a mAb
against human -actin (1:50, HHF-35; DAKO). HIF-1 was detected in
human skeletal muscle samples with a mAb against human HIF-1 (1:100,
Ab-4 clone H167; Neomarkers, Fremont, CA). This HIF-1 antibody
did not work on sections from rabbit tissues. Two antibodies from
different manufacturers were used for VEGF [1:500, clone sc-7269
(Santa Cruz Biotechnology, Santa Cruz, CA) and 1:200 (R&D Systems,
Minneapolis, MN)] and VEGFR-2 [1:500, clone sc-6251 (Santa Cruz
Biotechnology) and 1:200 (Research Diagnostics Inc., Flanders,
NJ)].29
In addition, the expression of VEGFR-1 (Flt-1)
was studied with a polyclonal antibody against human VEGFR-1 (1:500,
clone sc-316; Santa Cruz Biotechnology). Controls for immunostainings
included incubations with irrelevant class- and species-matched
immunoglobulins and incubations in which the primary antibody was
omitted. Photographs of histological sections were taken using an
Olympus AX70 microscope (Olympus Optical, Japan) with analySIS software
(Soft Imaging System GmbH, Germany). Immunostainings were graded as
follows: -, absent (0% of section area showed staining); +, weak (0
to 10%); ++, moderate (10 to 50%), or +++, strong immunostaining (50
to 100%).
In Situ Hybridization
The expression of VEGF and VEGFR-2 mRNA were studied in human and rabbit ischemic muscles by in situ hybridization either using radioactive [33P]UTP-labeled or nonradioactive digoxigenin-labeled riboprobes. A riboprobe covering the whole coding region of human VEGF165 cDNA and a 377-bp fragment of human VEGFR-2 cDNA were used with corresponding sense probes as controls as previously described.28 For nonradioactive in situ hybridization cDNAs were transcribed using the DIG RNA labeling kit (Boehringer Mannheim GmbH, Mannheim, Germany) to get digoxigenin-labeled run-off transcripts. Positive signal was then detected in paraffin-embedded sections using NBT/BCIP as the color substrate as described.30
Magnetic Resonance Imaging (MRI)
MRI was done for rabbits with hindlimb ischemia induced 1 or 6 weeks earlier using Varian UNITYINOVA (Varian Inc., Palo Alto, CA) imaging console interfaced to a 4.7 T horizontal magnet (Magnex Scientific Ltd., Abingdon, UK) with actively shielded gradients (Magnex Scientific Ltd.) and a custom-built surface coil (50 mm in diameter) placed between the thighs. MRI data were acquired using a flow compensated T2*-weighted three-dimensional gradient-echo sequence (FOV, 6 x 8 x 6 cm3; matrix, 256 x 128 x 64; TR, 25 ms; TE, 80 ms) with gadodiamide-contrast medium (GdDTPA-BMA; Omniscan, Nycomed, Norway). GdDTPA-BMA was injected via ear vein as a bolus (0.25 mmol/kg) and the acquisition was started 3 minutes after the injection. It has been previously shown that a GdDTPA-enhanced region of MRI encompasses both viable peri-infarction and nonviable infarction zones in myocardial ischemia.31
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In acute ischemia because of the occlusion of the femoral artery
or prosthesis (patients 1 and 2 in Table 1
) little VEGF
or VEGFR-2 and relatively normal muscle histology was found in the
proximal control samples (Figure 1
; a to
d). In the distal muscle samples histological sections revealed
thrombus formation in blood vessels and interstitial edema consistent
with acute ischemia and vascular permeability induced by strong VEGF
expression (Figure 1, e and f)
. By immunohistochemistry, VEGFR-2 was
also found in skeletal myocytes of the distal muscle samples (Figure 1g)
. VEGF immunostaining was mostly cytoplasmic whereas VEGFR-2 was
located on the cell membranes. As shown by CD31 immunostaining, a high
number of capillaries and large endothelial cell-covered vessels
without visible media or adventitia layers were observed in the same
areas (Figure 1f)
. Abundant nuclear HIF-1 immunostaining suggests
that the widespread VEGF expression in acute lower limb ischemia is
mediated by HIF-1 (Figure 1h)
.
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In patients with chronic critical lower-limb ischemia (patients
3–8 in Table 1
), VEGF protein and mRNA were found in atrophic
but not in normal skeletal muscle cells by immunohistochemistry (Figure 2c)
and in situ hybridization
(Figure 1, i and j)
. VEGFR-2 was also expressed in atrophic skeletal
muscle cells confirmed by two different monoclonal antibodies used
against VEGFR-2 (Figure 1m
and Figure 2d
) and in situ
hybridization (Figure 1, k and l)
. As a rule, the more atrophic
were the myocytes the stronger was the VEGF and VEGFR-2
expression. Adipose cell- and monocyte-macrophage infiltration was
associated with atrophic skeletal muscle cells (Figure 2b)
.
Furthermore, an increased number of blood vessels were co-localized
with regions of atrophic muscle and VEGF expression (Figure 2e)
. In
patients 3 and 4 there was a significant difference in the degree of
ischemia between control and ischemic samples both in the macroscopic
and microscopic examination, whereas in the samples from patients 5 to
8 only a small difference could be observed. Consequently, in patients
3 and 4 more VEGF immunoreactivity with more extensive atrophy was
observed in the distal samples compared to the proximal samples. In
addition to atrophic myocytes, VEGF expression was found in
regenerating human skeletal muscle cells of patients 5 to 7 with
chronic critical ischemia, especially in fibers where regeneration had
recently begun (Figure 1, n and o)
. Regenerating skeletal muscle fibers
can be distinguished from normal fibers by their small size and central
nuclei. HIF-1 was associated with VEGF expression in atrophic but
not with regenerating myocytes of chronically ischemic legs (Figure 1p)
. However, HIF-1 expression was diminished in atrophic myocytes
as compared to acutely ischemic muscle cells. Expression of VEGFR-1 was
restricted to endothelium both in acute and chronic skeletal muscle
ischemia (data not shown).
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Complete removal of femoral artery combined with the ligation of
lateral femoral circumflex and deep femoral arteries has been
frequently used as a model for hindlimb ischemia.27,32
The
procedure leads to a lack of arteries supplying major muscles of the
thigh, the quadriceps femoris and adductor muscles (Figure 3a)
. As a consequence, as shown by
GdDTPA-BMA-enhanced MRI 7 days after induction of ischemia,
extensive ischemic damages are often present in these muscles (Figure 3b)
. Six weeks after the removal of the femoral artery limb muscles
have regenerated because the collateral-dependent blood flow to the
limb has improved (Figure 3c)
. However, pathological changes and muscle
atrophy are still visible in MRI as indicated by extravasation of
GdDTPA-BMA (Figure 3c)
. Histological sections at the same time point
reveal sustained low VEGF expression and increased vascularization
indicating a role for VEGF in increasing capillary density and vascular
permeability (Figure 3, h and i)
.
|
. In animals killed on day 3 VEGF was
expressed diffusely throughout the affected muscles accompanied by
mononuclear cell infiltrates and necrotic skeletal muscle cells (Figure 3, d and e)
. However, as opposed to acute human skeletal muscle
ischemia, little VEGFR-2 was found in ischemic rabbit myocytes at this
time point. Both after 1 and 3 weeks of ischemia, regenerating myocytes
were the main source of VEGF (Figure 3f
and Figure 4a
). At 3
weeks fibrosis was also often present in the ischemic limb muscles. By
6 weeks regeneration had ended in most cases, and adipose tissue
infiltrate and fibrosis were observed. At this time point only
regenerated myofibers contained small amounts of cytoplasmic and
nuclear VEGF immunostaining (Figure 3h)
.
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One week after excision of the femoral artery, strong VEGF
expression was found in regenerating skeletal myocytes in ischemic
rabbit hindlimb muscles (Figure 4, a and e)
. Both VEGF immunohistochemistry and in situ hybridization
for VEGF mRNA gave similar results. According to immunohistochemistry
VEGF was mostly cytoplasmic, but also the nuclei of the regenerating
myotubes stained intensively (Figure 4a)
. VEGFR-2 was strongly
expressed in the same regenerating myotubes (Figure 4
; b and g).
Expression of both VEGF and VEGFR-2 was strictly restricted to
regenerating myocytes whereas regions with no regeneration were absent
of VEGF and VEGFR-2 (Figure 4, a and b)
. An increased number of
capillaries, which were also notably viable and dilated with
up-regulation of CD31, was associated with VEGF expression (Figure 4c)
.
Furthermore, macrophages had infiltrated into areas of regeneration
(Figure 4d)
. Similar to human samples, in sections where regeneration
had been recently begun, VEGF was present at the edges of myocytes
(Figure 4i)
where regeneration starts with satellite cell activation.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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expression has been studied in the
ischemic human myocardium.33
However, the expression
patterns of HIF-1, VEGF, and VEGFR-2 in ischemic human and animal
skeletal muscle have remained unclear. We demonstrate here two patterns
of VEGF and VEGFR-2 expression in skeletal muscle ischemia. In acute
ischemia VEGF and VEGFR-2 are expressed diffusely, VEGF originating
from muscle cells and macrophages and VEGFR-2 being present on the cell
membranes of ischemic myocytes. In chronic skeletal muscle ischemia and
in skeletal muscle recovering from ischemia VEGF and VEGFR-2
expressions are restricted to atrophic and regenerating muscle cells.
In both cases VEGF expression is co-localized with macrophage
infiltration and an increased number of blood vessels. Similarly with
VEGF and VEGFR-2 expression, HIF-1 expression was widespread in
acutely ischemic muscles but only atrophic myocytes expressed
detectable amounts of HIF-1 in chronically ischemic skeletal muscle.
On the other hand, HIF-1 was not associated with VEGF and VEGFR-2
expression in skeletal muscle regeneration. In accordance with our
results the increase in HIF-1 levels was shown to be transient
compared to more persistent VEGF expression in evolving myocardial
infarction.33
Furthermore, in vitro experiments
have shown that that there are HIF-1-independent signaling pathways
to induce VEGF expression.34
Thus, high level VEGF
expression in regenerating myotubes is probably because of the
activation of signaling pathways other than HIF-1. Previously expression of VEGF and its receptors has been shown to be up-regulated in response to various tissue injuries, but its role has been considered to be mainly restricted to angiogenesis and re-endothelialization of blood vessels.3,35,36 For example, after an acute phase of ischemia with diffuse expression of VEGF and its receptors, Li and colleagues36 demonstrated sustained expression of VEGF and VEGFR-2 in regions bordering the infarct zone in rat myocardium. After mechanical arterial injury VEGF and VEGFR-2 are up-regulated in arterial smooth muscle cells contributing to the repair of vascular endothelium.29,35 The present study shows for the first time that VEGFR-2 is expressed in skeletal myocytes. This novel finding suggests that the VEGF/VEGFR-2 system may have functions distinct from blood vessel formation in the survival and regeneration of skeletal muscle affected with ischemia. In addition, macrophages were co-localized with muscle atrophy, regeneration, and VEGF expression both in acutely and chronically ischemic skeletal muscle. Macrophages express VEGFR-137 and thus they are potentially recruited by VEGF in the regions of muscle regeneration and atrophy. In addition to their function in scavenging cell debris, macrophages play an essential role in triggering muscle regeneration, potentially through a variety of secreted cytokines and growth factors.38,39
Like VEGF in the current study, hepatocyte growth factor is expressed in a transient manner during postischemic muscle regeneration, with no expression in normal muscle.40 In fact, the similarities of hepatocyte growth factor with VEGF expression in skeletal muscle regeneration seem so striking that it is likely that there are common regulators of these growth factors in regenerating myocytes. It is also remarkable that VEGF, a secreted growth factor, and VEGFR-2 are found not only on the cell membrane but also in cytoplasm and nuclei of regenerating and atrophic skeletal muscle cells. Several lines of evidence now demonstrate that VEGF can be internalized after binding to VEGFR-2, and this ligand receptor complex travels to the nucleus leading to the up-regulation of VEGFR-2.41,42 Furthermore, the internalization of VEGF by VEGFR-2 results in phenotypical changes at least in endothelial cells.42,43 Interestingly, it has been shown that the activation of the phosphatidylinositol-3-OH-kinase/protein kinase Akt (PI3K/Akt) signal transduction pathway leads to the up-regulation of VEGF in an autocrine manner, induces myogenic differentiation and angiogenesis, and mediates the inhibitory effects of VEGF on apoptosis.5,44,45 Thus, an autocrine stimulation of the VEGF/VEGFR-2 system may have an important role in cell differentiation, such as in the conversion of myoblasts into myocytes in regenerating skeletal muscle. In chronically ischemic skeletal muscles, the anti-apoptotic properties of VEGF may contribute to the survival of muscle cells.
In the rabbit model, which is frequently used to study therapeutic effects of growth factors and genes in hindlimb ischemia, extensive ischemic damage and diffuse VEGF expression was detected in the thigh and leg muscles 3 days after induction of ischemia. After the acute phase abundant VEGF/VEGFR-2 expression occurred in regenerating myocytes as studied 1 to 6 weeks after surgery. Thus, it is possible that strong endogenous VEGF/VEGFR-2 expression and muscle necrosis in this model may confound the results of a therapeutic intervention, especially if the treatments are given shortly after surgery.
Animal and human data of this study suggest that VEGF and VEGFR-2
expression is attenuated in chronic skeletal muscle ischemia compared
to acute ischemia possibly because of diminished expression of
HIF-1. Excess VEGF provided by therapeutic means seems justified
especially in the regions of chronically ischemic limbs where
endogenous VEGF expression is low. Because VEGF is apparently involved
in skeletal muscle regeneration and survival, the administration of
exogenous VEGF by various approaches may be useful for the recovery of
ischemic skeletal muscle or myocardium both via angiogenesis-dependent
and -independent mechanisms. It is concluded that in acute ischemia
VEGF and VEGFR-2 are expressed diffusely whereas in chronic ischemia
only atrophic and regenerating muscle cells express VEGF and VEGFR-2.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, the Academy of Finland, the Orion Research Foundation, the Maud Kuistila Foundation, and Kuopio University Hospital (EVO grant no. 5130).
Accepted for publication January 10, 2002.
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