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Ann Thorac Surg 2007;84:43-49
© 2007 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

Interactive Effects of Homocysteine and Copper on Angiogenesis in Porcine Isolated Saphenous Vein

Nilima Shukla, PhD, Gianni D. Angelini, FRCS, Jamie Y. Jeremy, PhD*

Bristol Heart Institute, University of Bristol, Bristol, United Kingdom

Accepted for publication March 29, 2007.

* Address correspondence to Dr Jeremy, Bristol Heart Institute, Department of Cardiac Surgery, Bristol Royal Infirmary, Bristol, BS2 8HW, United Kingdom (Email: j.y.jeremy{at}bris.ac.uk).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: After coronary artery bypass grafting procedures with saphenous vein, there is a protracted elevation of plasma homocysteine and copper. These interact to elicit endothelial dysfunction through promotion of superoxide. It has been suggested that angiogenesis and the formation of a neovasa vasorum is important in mediating vein graft patency. A novel in vitro model of angiogenesis in isolated pig saphenous veins was therefore developed to study the effect of homocysteine and copper and the role of superoxide on tubule growth, an index of angiogenesis.

Methods: Two-millimeter rings of porcine saphenous veins were embedded in fibrin, incubated for 2 weeks with homocysteine and copper chloride, and tubules counted.

Results: Tubule growth in cultured saphenous veins, which was inhibited by angiostatin, occurred in a time-dependent manner during a 14-day period. Copper chloride alone at 1 µM and 10 µM augmented microtubule formation, whereas homocysteine alone at up to 1 mM had no effect. Homocysteine and copper chloride together markedly inhibited microtubule formation. Significant inhibition of tubule formation and superoxide formation was elicited with inhibitors of nicotinamide adenine dinucleotide phosphate oxidase, mitochondrial respiration, and xanthine oxidase. Copper chloride augmented superoxide formation, but homocysteine had no effect. Homocysteine and copper chloride together also augmented superoxide formation.

Conclusions: These data indicate that the increase in plasma homocysteine and copper may exert a deleterious effect on graft patency by preventing the formation of a neovasa vasorum, thereby promoting hypoxia. This effect is mediated by a mechanism independent of superoxide which actually promotes angiogenesis in this model.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Autologous saphenous vein continues to be the most widely used conduit for coronary artery bypass grafting (CABG) [1], and infrainguinal bypass operations for reconstruction of lower limb arteries [2]. However, as many as 50% of vein grafts fail within 10 years after the procedure [1, 2]. Vein graft failure involves the formation of a neointima [1–3]. Superimposed atherogenesis ultimately increases the risk of late graft failure [1–3]. The etiology of vein graft failure is still not fully clarified, and an effective therapeutic intervention, apart from aggressive lipid lowering [4], has not yet been implemented. Further studies on the etiology of vein graft disease are therefore required to devise effective interventional therapies.

It has been demonstrated that after CABG, there is a significant increase in the plasma concentrations of homocysteine, ceruloplasmin, and copper [5], which are three independent risk factors for cardiovascular disease [6–8]. Elevated plasma homocysteine is a risk factor for vein graft failure after infrainguinal bypass procedures [9] and CABG [10]. Although the consequences of this to vein graft patency is unknown, homocysteine and copper are known to interact to elicit endothelial dysfunction, in particular, the inhibition of nitric oxide (NO) formation through the generation of reactive oxygen species, including superoxide [11–13].

Surgical removal of the saphenous vein results in a loss of integrity of the vasa vasorum of the saphenous vein, which may promote tissue hypoxia [12, 14, 15]. Because graft thickening increases oxygen demand, which may further increase intragraft hypoxia [16, 17], the regrowth of a neovasa vasorum may constitute an important adaptation of saphenous vein grafts [15]. Indeed, external stenting of experimental vein grafts markedly inhibits neointima formation, an effect ascribed to the promotion of a neovasa vasorum [16]. It is possible, therefore, that the elevation of plasma copper and homocysteine may impair the repair and reintegration of the vasa vasorum. To test this hypothesis, the effect of homocysteine and copper on angiogenesis in the isolated pig saphenous vein was studied. The role of reactive oxygen species was also studied because homocysteine and copper have been shown to interact to generate superoxide [11].


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Tissue Collection and Preparation
Studies were performed on saphenous vein obtained from Landrace pigs (weight, 30 to 35 kg), which received humane care according to the Home Office Animals Care regulations. Anesthesia was induced with ketamine (30 mg) and atropine (0.6 mg), administered intramuscularly. A longitudinal incision was made on the outer aspect of the hind limb, and the saphenous vein removed, rinsed, and placed in cold Dulbecco Minimum Essential Medium (DMEM; Sigma Chemical Co, Poole, Dorset, United Kingdom [UK]).

In Vitro Assay of Angiogenesis
In vitro tubule formation, an index of angiogenesis by intact blood vessels, was assessed by using established methods [18, 19]. Adventitia was removed from the saphenous veins, and 2-mm rings were prepared with a scalpel. Fibrinogen solution (3 mg/mL; Sigma Chemical Co) and thrombin solution (50 U/mL; Sigma Chemical Co), which together form fibrin [19], were added to each well, thereby creating a fibrin "bed" (Fig 1). Each ring was then placed on the fibrin bed, and fibrinogen and thrombin were again added, embedding and immobilizing the rings. The fibrin-ring complex was then covered with serum-free DMEM containing 100 µg/mL aminocaproic acid (Sigma Chemical Co) [19].


Figure 1
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  		Fig 1. Diagrammatic representation model of angiogenesis in the pig saphenous vein: (a) thrombin plus fibrinogen are placed in the bottom of the well, creating a fibrin bed; (b) rings of saphenous vein are placed on the fibrin bed, and thrombin plus fibrinogen are placed over the ring, effectively embedding the ring in fibrin; (c) fibrin is covered with serum free medium, and (d) tubules grow into fibrin which are then counted over time.

 
The culture plates were then placed in a 95% oxygen/5% carbon dioxide humidified incubator for up to 14 days. Culture medium was changed every 2 days. To assess tubule formation, photographs were taken each day by using a phase-contrast microscope, and tubules were counted by a technician blinded to treatments. Tissues at different time points were placed in 4% paraformaldehyde solution for histologic appraisal or flash frozen for immunocytochemical analysis.

To validate the model, the effect of angiostatin and thapsigargin (Sigma), two known inhibitors of angiogenesis [17, 19] on tubule formation and growth, was assessed as described. For studies on the effect of homocysteine and copper, rings were embedded as described and incubated with DMEM containing homocysteine and copper chloride at concentrations found in the plasma of patients who have undergone CABG (between 1 and 10 µM for copper and 10 and 100 µM for homocysteine) [5].

For studies on reactive oxygen species, explants were preincubated with diphenyleneiodonium chloride (DPI) and apocynin (nicotinamide adenine dinucleotide phosphate [NADPH] oxidase inhibitors), rotenone (an inhibitor of mitochondrial respiration), and allopurinol (an inhibitor of xanthine oxidase). The role of NO was assessed by incubating explants with N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase [22]. Sigma Chemical Co supplied all of these substances.

Measurement of Superoxide Formation
Saphenous vein rings were placed in individual wells of a 24-well plate and equilibrated in DMEM (without phenol red) for 10 minutes at 37°C in a 95% air-5% carbon dioxide incubator (Heraeus, Hera Cell, Kandro Laboratory Products, Germany) [12, 22]. Cytochrome c (20 µM), with or without 500 U/mL copper-zinc superoxide dismutase, was added and incubated at 37°C in a 95% air-5% carbon dioxide incubator for 1 hour. The reaction medium was removed and reduction of cytochrome c determined at 550 nm in an Anthos Lucy 1 spectrometer (Lab-tech International, Ringmer, East Sussex, UK) and converted to moles of superoxide, using {Delta}E550 nm = 21.1 mM/cm as the extinction coefficient. The reduction of cytochrome c that was inhibitable with SOD reflected actual superoxide release. Segments were rinsed in phosphate-buffered saline (PBS), and weighed. Data are expressed as micromoles of superoxide per milligram of tissue per hour.

Immunocytochemistry
Endogenous peroxidase activity was inhibited by incubation at room temperature in 3% hydrogen peroxide for 10 minutes. The sections were placed in a humidified chamber and incubated with neat goat serum for a further 30 minutes at room temperature. For endothelial nitric oxide synthase (eNOS), sections were incubated at 1:200 with mouse monoclonal anti-eNOS (BD Biosciences, Cowley, Oxford, UK) overnight in a humidified chamber at 4°C. For lectin staining, sections were rinsed in PBS, and free avidin sites were blocked with a biotin blocking kit (Vector, Burlingame, Canada). Neat goat serum was added and incubated for 30 minutes and then with biotinylated lectin from Dolichos biflorus agglutinin (diluted 1:500) overnight at 4°C, followed by incubation for 30 minutes with horseradish peroxidase-conjugated extravidin (1:200; Burlingame).

Sections were then incubated with secondary antibody, biotinylated goat antimouse immunoglobulin G antibody diluted 1:400 in PBS and 1% bovine serum albumin at room temperature for 30 minutes. Sections were then incubated with extravidin horseradish peroxidase diluted 1:200 in PBS and 1% BSA for 30 min, washed and incubated for 10 minutes with N,N-dimethyl-4-aminoazobenzene at room temperature to allow for color development. Sections were counterstained with hematoxylin (Sigma) for 1 minute and dehydrated with ethanol and HistoClear (National Diagnostics, Atlanta, GA) and sections mounted on slides.

Data Analysis and Statistics
All measurements represent the mean of means. Quadruplicate measurements were made for each parameter per data point and the mean was calculated. In turn, for each parameter, measurements were made in tissue from 6 separate pig saphenous veins. Data analysis was done by using GraphPad Prism 4 software (GraphPad Software, San Diego, CA). The Kruskal-Wallis test was used to establish that the data were all normally distributed, and as such, parametric statistics was deemed applicable. Data are expressed as mean ± standard error of the mean. Data were analyzed using analysis of variance for multiple comparisons. Paired comparisons between two groups were performed using the paired Student t test when analysis of variance indicated statistical significance for the multiple comparison. Statistical significance was accepted at p < 0.005.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Tubule growth in cultured saphenous vein explants embedded in fibrin occurred in a time-dependent manner during a 14-day period (Fig 2, Fig 3). Serum was not required because the tissue explant deploys nutrients from its own endogenous stores. A trigger (ie, serum) for angiogenesis (as for replication) is also not required because angiogenesis is clearly robust under these conditions. We have done experiments with serum, and more growth is not seen. However, serum contains so many proangiogenic factors, including copper and homocysteine, that this might be a confounding factor and would mask effects. The growth of microtubules was inhibited by 1 µg/mL angiostatin and 10 nM thapsigargin, again during a 14-day incubation of saphenous vein rings (Fig 2, Fig 3).


Figure 2
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  		Fig 2. Tubule formation by porcine saphenous vein rings embedded and incubated in fibrin over time: control (bullet); 1 µg/mL angiostatin ({blacktriangleup}); 10 nM thapsigargin ({diamondsuit}); 1 µM copper chloride ({blacksquare}); 10 µM copper chloride ({circ}). (See also Figures 3 and 5.) Each point = mean ± standard error of the mean; n = 6 saphenous vein rings. *p < 0.01 comparing effect at individual time points with controls (untreated).

 

Figure 3
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  		Fig 3. Representative photomicrographs demonstrate tubule formation in porcine saphenous vein explants assessed at 7 days after embedding in fibrin: (a) control, (b) 10 nM thapsigargin, (c) 1 µg/mL angiostatin (see Fig 2).

 
Immunocytohistochemistry demonstrated positive staining for endothelial cells and eNOS, which were located at the luminal surface and at the outer adventitial region (Fig 4). Copper alone, at 1 and 10 µM, augmented the rate of microtubule formation (Fig 5, Fig 6). At 100 µM and 1 mM, however, homocysteine alone had no significant effect (Fig 5). By contrast, homocysteine and copper together, at concentrations found in the plasma of patients who have undergone CABG, significantly inhibited tubule formation (Fig 5, Fig 6). The copper chelator, penicillamine also inhibited microtubule formation in a dose-dependent manner in the absence of exogenous copper (Table 1).


Figure 4
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  		Fig 4. Immunocytochemical staining of (left) lectin (endothelial cells), (right) endothelial nitric oxide synthase (eNOS) in pig saphenous veins Positive staining indicated by large arrows. Note staining is at the lumen endothelium and on the outer surface (adventitial) of the medial layer.

 

Figure 5
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  		Fig 5. Effect of homocysteine at 100 µM ({blacksquare}) and 1 mM ({circ}) on tubule formation in porcine saphenous vein explants compared to controls (bullet) and the effect of 1 µM copper chloride + 10 µM homocysteine; ({blacktriangleup}) and 10 µM copper chloride + 100 µM homocysteine over a 14 day time course. Each point = mean ± standard error of the mean; n = 6 experiments. *p < 0.01 comparing effect at individual time points with controls (untreated).

 

Figure 6
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  		Fig 6. Representative photomicrographs demonstrate tubule formation assessed at 7 days after embedding in fibrin. (A) Control, (B) 1 µM copper chloride, (C) 10 µM copper chloride, (D) 10 µM homocysteine plus 1µM copper chloride, (E) 100 µM homocysteine plus 1 µM copper chloride; (F) 100 µM homocysteine + 10 µM copper chloride. (Bar = 500 µm.)

 

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  		Table 1 Effect of Penicillamine on Tubule Formation by Isolated Pig Saphenous Vein Rings Assessed at 7 Days After Being Embedded in Fibrin
 
Significant inhibition of tubule formation and superoxide formation was observed with DPI (10 µM), apocynin (10 µM), rotenone (100 µM), allopurinol (100 µM), and L-NAME (500 µM; Table 2). Copper augmented superoxide formation, homocysteine had no effect, and copper and homocysteine together also augmented superoxide formation (Table 3).


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  		Table 2 Effects on Tubule Formation and Superoxide Formation in Saphenous Vein Rings at Day 7 After Being Embedded in Fibrin
 

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  		Table 3 Effect of Copper Chloride and Homocysteine, Alone and Together, on Superoxide Formation by Isolated Pig Saphenous Vein Rings Assessed at 7 Days After Being Embedded in Fibrin
 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
The first objective of this study was to devise an in vitro means of assessing angiogenesis in the isolated saphenous vein. Thus, when embedded in fibrin (a potent angiogenic factor in its own right) [17] and incubated for up to 14 days, microtubules grew into the fibrin in a time-dependent manner. It is notable that external stents and sheaths elicit a marked promotion of microvessel formation, an effect ascribed to the accumulation of fibrin in the graft–stent interface [16]. In the present study, angiogenesis was blocked by angiostatin, a standard specific inhibitor of angiogenesis [17], and by thapsigargin, which blocks microtubule formation through depletion of calcium pools [19], thus consolidating the validity of the model. Immunocytochemical appraisal of the saphenous veins also demonstrated positive staining for endothelial cells (lectin) and eNOS, which were located at the luminal surface, as one would expect, but also at the outer medial/adventitial region.

This indicates that even though the adventitia has been removed, there are sufficient microvessels at the outer surface to allow for the regeneration of microvessels (and neovasa vasorum) through de novo angiogenesis. Surgical preparation of saphenous veins for CABG also entails removal of the adventitia.

The present study demonstrates that exogenous copper potentiates tubule formation in isolated saphenous vein explants. This is in accord with the well-established proangiogenic effect of copper [20]. Other studies have demonstrated that copper promotes proliferation of isolated endothelial cells [21], promotes angiogenesis in a wound-healing model [22], the expression of vascular endothelial growth factor (VEGF) [23], and the activation of lysyl oxidase [24]. Penicillamine, a copper chelator, inhibited tubule formation, consolidating that that endogenous copper plays a role in modulating angiogenesis in the isolated saphenous vein.

By contrast, homocysteine alone, even at concentrations much greater (up to 1 mM) than those found in plasma after surgical procedures [5] and in patients with peripheral arterial disease [13], had no effect on microtubule growth by isolated saphenous vein. However, combinations of homocysteine and copper as low as 1 and 10 µmol/L, respectively, which are similar to concentrations found in postsurgical CABG patients (ie, plasma homocysteine between 10.1 and 13.5 µmol/L and plasma copper between 13.5 and 20 µmol/L) [5], elicited a marked inhibitory effect. At physiologic concentrations, homocysteine and copper have already been shown to interact to inhibit NO formation [11]. Endothelial regrowth in the carotid artery of hyperhomocysteinemic mice is impaired [25]. Furthermore, the administration penicillamine, a copper chelator, reverses the inhibitory effect of hyperhomocysteinemia on NO formation [12]. Copper and homocysteine also interact to promote the oxidation of low-density lipoprotein and vascular cell damage [26, 27].

Homocysteine and copper are also known to interact chemically to generate superoxide, which in turn can negate NO bioactivity through reactions that form nitrogen species, such as peroxynitrite [28, 29]. Because NO promotes angiogenesis [17], it is possible that the inhibition of angiogenesis by homocysteine and copper may be due to the overproduction of superoxide. In the present study, however, inhibitors of NADPH oxidase, xanthine oxidase, and mitochondrial respiration [30] all inhibited tubule formation. This indicates that superoxide promotes angiogenesis rather then impairs it, which is consistent with other reports [31]. Copper alone also promoted superoxide formation, whereas homocysteine and copper together had little effect in the present study and as such cannot account for their combined inhibitory effect on tubule formation. Homocysteine and copper also form a complex that exerts biologic effects independently of superoxide. These include the inhibition of activation of protein kinase C and mitogen activated protein kinase, decreased glutathione peroxidase activity and alterations of focal adhesion complexes [32–34].

From a pathophysiologic perspective, the sustained increase in plasma homocysteine and copper (6 weeks after operation) coincides with the rapid (within 1 month) adaptive thickening of vein grafts and, more important, neointima formation. It is a widely held view that inhibition of neointima formation at this early stage ultimately prevents vein graft failure in the longer term (ie, during the ensuing 10 years). The elevated plasma levels of copper and homocysteine associated with CABG may therefore inhibit adaptive angiogenesis and promote hypoxia within recently implanted vein grafts in this crucial period. Furthermore, because the endothelial regrowth and relining of the conduit is considered a possible determinant of vein graft patency [35] and involves the same mechanisms as angiogenesis, endothelial repair may also be compromised by elevated copper and homocysteine.

Therapeutically, there are two means by which this interaction could be reduced. First, folic acid, which reduces plasma homocysteine, improves endothelial function [36]. Folic acid and its active metabolite, 5-methyltretrahydrofolate, also have direct effects that conserve the integrity of endothelial NO formation [37], and as stressed, NO is axiomatic in promoting angiogenesis. Folic acid has the additional advantage of being relatively free of side effects, and as such, long-term use may be beneficial in reducing atherogenic graft disease. One could also prevent the interaction with a copper chelator. Indeed, thiomolybdate, a copper chelator, has proven effective in reducing inflammation [38] and to have minimal side effects. Because copper chelators inhibit angiogenesis, however, this may preclude their therapeutic usefulness in vein graft patients.

The present model represents a reliable means of assessing potential therapies aimed at promoting angiogenesis, including gene transfer, which can be used to determine their efficacy before the more expensive in vivo experimentation, as well the fundamental biology of microvessel repair in vein grafts. It may seem preferable to use human saphenous veins, but the innate drawback is the considerable heterogeneity of patients, which impairs reproducibility of responses. The next step, however, is to develop the model for use in human saphenous veins.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This research was supported by a grant from the British Heart Foundation.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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