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Ann Thorac Surg 2000;69:1750-1754
© 2000 The Society of Thoracic Surgeons

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Original articles: Cardiovascular

Differences in myocardial and peripheral VEGF and KDR levels after acute ischemia

^a Cardiothoracic Research Laboratories, East Carolina University School of Medicine, Greenville, North Carolina, USA
^b Department of Surgery, East Carolina University School of Medicine, Greenville, North Carolina, USA
^c Department of Physiology, East Carolina University School of Medicine, Greenville, North Carolina, USA

Address reprint requests to Dr Francalancia, Division of Cardiothoracic Surgery, Department of Surgery, East Carolina University School of Medicine, Greenville, NC 27854
e-mail: nfrancalancia{at}brody.med.ecu.edu

Presented at the Forty-sixth Annual Meeting of the Southern Thoracic Surgical Association, San Juan, Puerto Rico, November 4–6, 1999.

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Recent clinical use of vascular endothelial growth factor (VEGF) in the treatment of both myocardial and peripheral ischemia has suggested the possibility of tissue specific coregulation of VEGF and its receptors (eg, kinase domain region [KDR]). The present study was performed to detect the relationship between VEGF and KDR protein levels after acute myocardial and peripheral ischemia.

Methods. Eleven dogs were divided into two groups: peripheral ischemia (n = 6, ligation of major limb arteries) and myocardial ischemia (n = 5, circumflex artery ligation). Muscle biopsy specimens were taken from the perfusion territories of the occluded circumflex artery and limb arteries 3 hours and 6 hours after ligation. Protein levels were determined using Western blot analysis.

Results. In myocardium, VEGF levels increased on average eightfold from baseline (p < 0.05) both 3 hours and 6 hours after occlusion, whereas myocardial KDR levels dropped by about 60% at 3 hours and 80% at 6 hours (p < 0.05). With limb ischemia, both VEGF and KDR levels were significantly elevated at 3 hours.

Conclusions. In acute ischemia, regulation of VEGF and KDR may be controlled differently in cardiac and skeletal muscle. Myocardial KDR levels showed a significant decrease from baseline compared with a significant rise with peripheral ischemia.

	Introduction

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Therapeutic angiogenesis is the induction and acceleration of neovascularization [1], which has great potential to expand the scope of treatment of myocardial and peripheral vascular ischemic diseases. To evaluate its feasibility and safety, several groups have embarked on and completed phase I clinical trials involving growth factors [2–7] and other inducers of angiogenesis [8, 9]. However, many questions remain unresolved, particularly with respect to the method of delivery of the growth factors: protein versus DNA (deoxyribonucleic acid), percutaneous versus open administration, sole or adjuvant treatment. In addition, as all of the factors tested are expressed endogenously in response to ischemia, the mechanism of benefit associated with exogenous administration remains unclear. This is in part due to an incomplete but evolving knowledge of the regulation of angiogenesis.

The purpose of this study was to address possible differences in angiogenic regulatory mechanisms in skeletal and myocardial tissues. To this end, changes in levels of vascular endothelial growth factor (VEGF) (the most widely studied angiogenic growth factor) and kinase domain region (KDR), a receptor for VEGF that is responsible for almost all of its mitogenic activity [10, 11], were determined after acute local ischemia in skeletal muscle and myocardium.

	Material and methods

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Twelve mongrel dogs of either sex and weighing 18 to 22 kg were used. Anesthesia was induced using Surital (thiamylal sodium) (20 to 25 mg/kg intravenously) and was maintained using fentanyl (loading dose, 30 µg/kg intravenously; infusion, 0.4 µg · kg^-1 · min^-1) and midazolam hydrochloride (loading dose, 100 µg/kg intravenously; infusion, 0.6 µg · kg^-1 · L^-1). Intubation and mechanical ventilation were accomplished to establish baseline ventilatory values (arterial oxygen tension, 95 mm Hg, 35 mm Hg arterial carbon dioxide tension 42 mm Hg, and 7.36 pH 7.43) and were maintained throughout the procedure. All animals underwent continuous electrocardiographic and blood pressure (Millar transducer, Millar instruments; Millar Instruments, Inc, Houston, TX) monitoring.

Surgical protocol
Animals were initially divided into two groups of 6: a peripheral ischemia group that underwent ligation of major limb arteries (femoral and axillary) and a myocardial ischemia group that underwent ligation of the circumflex artery (xA). The experiments were conducted under a protocol approved by the institutional animal care and use committee. All animals in this study received humane care in compliance with East Carolina University and National Institutes of Health guidelines.

In both groups, the animals were placed in a supine position. In the peripheral ischemia group, small incisions were made over the proximal femoral and axillary arteries bilaterally. These four major limb arteries were ligated simultaneously. Muscle biopsy samples were obtained from individual limbs using a randomization schedule: prior to ligation (baseline) and 3 hours and 6 hours after ligation. The biopsy specimens were taken from skeletal muscle immediately adjacent to the site of ligation (proximal biopsy), from the muscle group at stifle (knee)/elbow joint (middle biopsy), and from the muscle group around the hock (ankle)/carpus (wrist) joint (distal biopsy).

In the myocardial ischemia group, all the animals underwent median sternotomy and isolation and ligation of the proximal CxA. After placement of instruments, each animal was assigned to either the 3-hour or 6-hour ligation subgroup. In the 3-hour subgroup, myocardial punch biopsy specimens (full thickness) were obtained 3 hours after ligation of the CxA and in the 6-hour subgroup, 6 hours after CxA ligation. The biopsy samples were taken adjacent to the ligation site and at two more locations progressively distal to the ligation. One of the dogs in the 3-hour subgroup went into persistent ventricular fibrillation shortly after CxA ligation and died.

All biopsy specimens were weighed and snap frozen in liquid nitrogen. At the completion of the study, all animals in both groups were killed by a lethal overdose of anesthesia.

Tissue analysis
Frozen biopsy samples were weighed, and homogenization buffer (20 mmol/L Trizma Base, 5 mmol/L EDTA [ethylene diamine tetraacetic acid], 2 mmol/L EGTA [egtazic acid], 10 mmol/L Benzamidine, 195 µL 2-mercaptoethanol, 1% Triton X-100, pH 7.4) plus protease inhibitors (1 mg/mL of leupeptin, 0.1 mg/mL of aprotinin, 0.1 mg/mL of ovalbumin, 0.1 mg/mL of phenyl-methyl sulfonyl-fluoride (PMSF) in ethyl alcohol (EtOH) was added in a tissue to buffer ratio of 1:0.5 (weight per weight). Tissue was homogenized with a Biospecs Products model 985-370 polytron set at 6. Homogenate was centrifuged at 10,000 g and 4°C for 20 minutes. Supernatant (soluble fraction) was removed and saved for ultracentrifugation. Whole-cell solubilized fraction was sonicated for 15 seconds. Samples were centrifuged at 100,000 g and 4°C for 30 minutes with a Beckman model L8-70M ultracentrifuge. Supernatant was saved for determination of protein concentration and Western blot analysis.

Protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Philadelphia, PA) based on the Bradford method. The spectrophotometer was set at a wavelength of 595 nm. Samples were prepared for Western blot analysis by adding 4x Western stop solution (8% SDS [sodium dodecyl sulfate], 240 mmol/L Trizma Base, 400 mmol/L Dithiothreitol, 0.04% bromophenol blue, 30% glycerol, pH 6.8) and 20 mmol/L Trizma Base to 200 µL of homogenate to give a final concentration of 1 to 10 µg/µL. Eight-percent polyacrylamide gels were loaded with samples of 100 µg of total protein. Gels were subjected to electrophoresis for 50 minutes at 200 V and 300 mA in running buffer (25 mmol/L Trizma Base, 192 mmol/L glycine, 0.1% SDS). Protein was electrophoretically transferred to nitrocellulose (0.45 µm) in a Bio-Rad Mini-PROTEAN II transfer system operating at 100 V for 3 hours.

Blots were blocked by 2-hour incubation in 5% evaporated milk in wash buffer (10 mmol/L Trizma Base, 100 mmol/L NaCl, 0.05% Tween 20, pH 7.5) at room temperature. They then were incubated in primary antibody (polyclonal anti-rabbit (sc-153) for VEGF and sc-6251 for KDR; Santa Cruz Biotechnology, Inc, Santa Cruz, CA) to specific isozyme at 1:5,000 dilution overnight (16 to 18 hours) at 4°C with agitation. Blots were washed for 30 minutes with wash buffer changed every 5 minutes. Blots were next incubated in anti-rabbit or mouse IgG-HRP conjugated antibody (Santa Cruz Biotechnology) at room temperature and agitated (1:2,500 dilution for the secondary antibody). The washing procedure was repeated in preparation for enhanced chemiluminescence detection. Protein bands were demonstrated using an ECL detection kit (Amersham, Rochester, NY), and Hyperfilm MP (Amersham) was exposed to blots for various periods to optimize the images. Images were scanned by Deskscan II software (Hewlett-Packard, Boise, ID), and the highlighted bands were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The optical densities were used for data reporting.

Baseline canine myocardial biopsy samples were obtained from nontreated animals. Control speciman was rat myocardium. On each of the polyacrylamide gels, two positive "control" lanes were loaded with rat myocardial protein (previously tested for the presence of VEGF and KDR). The densities of canine bands were later normalized to these controls to standardize quantitative comparison between gels.

Statistical analysis
All data are presented as the mean ± the standard error. Differences between groups were tested using Student’s t test. Changes within groups were analyzed using one-way analysis of variance, Mann-Whitney test (for comparing two groups), and Kruskal-Wallace test (for comparing more than two groups). A p value of less than 0.05 was considered significant.

	Results

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Peripheral ischemia group
The data obtained were initially evaluated with reference to each biopsy site. The changes seen were essentially the same for all three locations (proximal, middle, distal). The KDR levels were increased from baseline 3 hours after ligation and returned to baseline at 6 hours after ligation. The VEGF levels at 3 hours after ligation were increased (middle and distal sites); by 6 hours after ligation, a decrease toward baseline was noted. Because of the similarities in protein level variations, the decision was made to pool the data. Figure 1 shows temporal variations in KDR and VEGF levels, without differentiating by location. The same general trend is apparent: by 3 hours after ligation, an increase from baseline in both KDR (p < 0.05) and VEGF (p = 0.06) levels was shown. Levels of both returned toward baseline by 6 hours after ligation.

View larger version (21K): [in this window] [in a new window]   Fig 1. Peripheral ischemia group. Peripheral skeletal muscle protein levels of vascular endothelial growth factor (VEGF) and its receptor kinase domain region (KDR) after acute local ischemia. There was an increase in both levels with time; however, KDR returned to baseline (Base = Baseline; ADU = arbitrary densinometric units; * = p < 0.05 versus baseline).

View larger version (19K): [in this window] [in a new window]   Fig 2. Myocardial ischemia group. Protein levels of vascular endothelial growth factor (VEGF) and its receptor kinase domain region (KDR) obtained from the myocardium neighboring the circumflex artery after its acute ligation. Data consistently showed a drop in KDR values and an elevation in VEGF levels after ligation (Base = Baseline; ADU = arbitrary densinometric units; * = p < 0.05 versus baseline).

	Comment

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Angiogenesis normally occurs at variable rates in different organs, as it depends on the tissue regeneration. This response, however, is amplified under conditions that result in cellular hypoxia (ie, ischemia) [12–16]. Increased expression of angiogenic factors (especially VEGF) and their receptors is the next step. This leads to enrichment of interstitial fluid with plasma fluids because of increased vascular permeability caused by VEGF. Endothelial cells migrate into the rich medium, proliferate, and form structures that later will reshape into functional vessels, a process that has been fairly well studied [12–16]. In contrast, most of the recent basic science research has been focused on the cellular and intracellular changes resulting from KDR activation.

On binding to KDR, VEGF induces tyrosine autophosphorylation of KDR. This in turn leads to potent activation of mitogen-activated protein kinases in a time- and concentration-dependent fashion. In particular, the activities of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase have been shown to be essential for VEGF. The result of this cascade is induction of nuclear events such as transcription and entry into the synthesis phase of the cell cycle [17–19].

One of the benefits of understanding the basics of VEGF and KDR interaction and its intracellular cascade is the potential to find ways to alter or manipulate these mechanisms to the benefit of patients undergoing therapeutic angiogenesis. More importantly, such studies can potentially clarify why endogenous angiogenesis is inadequate in some patients despite clinically remarkable ischemia. For example, glypican-1, a family of glycophosphatidylinositol anchored cell surface heparan sulfate proteoglycans, has been identified as an extracellular chaperone that can restore the binding ability of VEGF when previously damaged by oxidation (caused by oxygen radicals released during wound healing) [20]. Glypican-1 could potentially decrease the needed dose of VEGF. Alternatively, it has been found that VEGF mRNA (messenger ribonucleic acid) is labile under normal oxygen level conditions. However, hypoxia induces combination of the destabilizing elements in the mRNA 3' and 5' terminus as well as their coding region [21]. Rather than administration of growth factors, this stabilizing mechanism could become a target for therapeutic angiogenesis. Finally and more directly, studies laying the groundwork for the design of VEGF analogues and mimics [22] have been performed.

However, it may be misleading to assume that VEGF signaling follows similar patterns in all ischemic tissues, and lessons learned in limb ischemia may not translate directly to myocardial ischemia.

The results of the present study demonstrate major differences in skeletal and myocardial response to local acute ischemia. The myocardial KDR protein levels decreased significantly, and VEGF protein levels were upregulated. These changes were sustained throughout the ischemic period. Alternatively, skeletal muscle KDR levels increased compared with baseline after acute ischemia, and there was a concomitant increase in skeletal muscle VEGF levels. However, protein levels reverted toward baseline by 6 hours after ligation.

The observed changes in KDR are noteworthy especially in view of previously published data showing upregulation of KDR mRNA in response to hypoxia [13]. This could partially be due to our emphasis on protein level and not mRNA; however, upregulation of mRNA would be expected to increase protein levels (keeping in mind that our results reflect whole-cell protein levels). Does protein follow message (as in skeletal muscle), or is protein contrary to message, as would be predicted for myocardium? If so, this suggests that transcription and translation of myocardial KDR protein are temporally divergent. This may be important. There also is evidence that inflammation and cytokines could alter VEGF and KDR levels [10, 11]. However, recent evidence suggests that VEGF induces a response in endothelial cells that is largely distinct from inflammatory stimuli [23].

Further studies investigating the effects of ischemic myocardium on nonischemic skeletal muscle and of ischemic muscle on nonischemic myocardium will be valuable in understanding the potential "cross-talk" in vascular tissue responses. These studies will help to optimize benefits and clarify the potential risks associated with various forms of therapeutic angiogenesis.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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