Oxidative stress and lipid retention in vascular grafts: comparison between venous and arterial conduits.

BACKGROUND: Because saphenous vein grafts (SVGs) exhibit greater cellular heterogeneity and worse clinical outcomes than arterial grafts (AGs), we examined oxidative stress and lipid retention in different vascular conduits. METHODS AND RESULTS: In a porcine model of graft interposition into carotid artery, superoxide anion (.O(2)(-)) was measured at 2 weeks after surgery. SVGs demonstrated increased.O(2)(-) production compared with AGs (SOD-inhibitable nitro blue tetrazolium reduction, P<0.01). The NAD(P)H oxidase inhibitor diphenyleneiodonium (P<0.01) abolished SVG-derived.O(2)(-), whereas the inhibitors of other pro-oxidant enzymes were ineffective. The change in oxidative stress was also reflected by lower activity of the endogenous antioxidant superoxide dismutase in SVGs than in AGs (P<0.001). SVG remodeling was associated with increased synthesis of sulfated glycosaminoglycans and augmented expression of a core protein, versican. These changes were accompanied by SVGs retaining significantly more (125)I-labeled LDL than AGs ex vivo (P<0.001). In hyperlipemic animals, lipid accumulation and oxidized epitopes were preferentially noted in the intima of SVGs at 1 month after surgery. CONCLUSIONS: This study demonstrated significant differences in the biology of SVGs and AGS: SVGs exhibited higher oxidative stress, LDL accumulation, and the presence of oxidized epitopes. These findings suggest that proatherogenic changes in SVGs may commence early after surgical revascularization.

Contractile smooth muscle cell apoptosis early after saphenous vein grafting.

BACKGROUND: The media of saphenous veins is composed of two cell populations: smooth muscle (SMC) and non-smooth muscle (NSMC) cells. Previous studies demonstrate a loss of SMCs by 3 days after grafting, despite an increase in cell proliferation. The purpose of this study is to determine the rates of apoptotic cell death versus cell proliferation for the two major cell populations of the media. METHODS: Veins (six/time point) were examined at 0.5, 1, 2, 4, 8, 24, and 48 hours after grafting in crossbred pigs. Terminal transferase-mediated dUTP nick end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) stains were used to assess apoptosis and proliferation. Apoptosis was also corroborated with confocal and electron microscopy. RESULTS: Apoptosis was high in both cell populations: at 8 hours, SMC and NSMC apoptosis peaked at 14.5% +/- 3.5% and 49.9% +/- 7.8%, respectively. In contrast, cell proliferation was different between the two populations. SMC proliferation was low at all time points, whereas NSMC proliferation rose to 22% +/- 5.4% by 48 hours. CONCLUSIONS: Medial SMCs are removed through apoptosis and appear to be replaced by fibrous tissue (NSMCs) early after vein grafting. This reciprocal change between the medial SMC and NSMC populations may contribute to late vein graft degeneration.

Sustained reduction of neointima with c-myc antisense oligonucleotides in saphenous vein grafts.

BACKGROUND: Treatment of saphenous veins with c-myc antisense oligomers during preparation for grafting reduces medial cellular proliferation and macrophage infiltration, and preserves medial smooth muscle content at 3 days. Accordingly, the purpose of this study was to examine whether c-myc antisense oligomers have an impact on late vein graft remodeling. METHODS: Sixty-two pigs underwent unilateral saphenous vein-carotid artery interposition grafting. Harvested veins were incubated either in saline (control group) or 20-micromol/L or 200-micromol/L concentrations of c-myc antisense oligomers (treated groups) for 30 minutes intraoperatively. Three months after surgery, vein graft histology was assessed. RESULTS: Forty-five of 62 randomized animals survived the experiment; no differences in animal survival or graft patency among the groups were observed (p = NS, chi2). C-myc antisense oligomers significantly decreased neointimal and wall thickness, as well as increased lumenal index, in treated groups (p<0.04, p<0.03, and p<0.001, respectively, analysis of variance). In contrast, there was no difference in medial thickness or perivascular wound healing. CONCLUSION: Intraoperative treatment of saphenous veins with c-myc antisense oligomers decreased neointimal formation at 3 months after grafting. In conjunction with our previous reports, these findings suggest that early inhibition of cellular proliferation and inflammatory infiltration results in a sustained reduction in neointimal formation and favorable graft remodeling.

Heparin and basic fibroblast growth factor are associated with preservation of latissimus cardiomyoplasties in goats: a retrospective study.

BACKGROUND: Chronic electrical stimulation of cardiomyoplasties often leads to atrophy and fibrosis of the skeletal muscle. In this retrospective study, we re-examined the data in our previous work, which suggested that muscle was preserved by treatment with basic fibroblast growth factor (bFGF). METHODS: Histologic sections were reviewed for evidence of atrophy, and fibrosis from four groups of goats with latissimus dorsi cardiomyoplasty: (1) unstimulated; (2) 2-Hz stimulated x 6 weeks; (3) 2-Hz stimulated with heparin infusion (50 units/hour) x 6 weeks; and (4) 2-Hz stimulated with bFGF (80-micrograms bolus/week) x 6 weeks. RESULTS: Muscle degeneration, as indicated by fat replacement of muscle fibers, was 56.95% +/- 9.16% (mean +/- S.E.) in the 2-Hz stimulated compared with 16.43% +/- 6.22% in unstimulated muscles. In 2-Hz = bFGF and 2 Hz-Heparin (Hep) groups, degeneration was 11.60% +/- 3.04% and 20.36% +/- 5.03%, respectively. bFGF treatment was associated with a greater latissimus blood flow than in the 2-Hz-untreated and 2 Hz-Hep groups (p < 0.05). CONCLUSIONS: bFGF's protection against degeneration may have involved angiogenesis and myogenesis, whereas that of heparin appears to have involved only myogenesis. While the mechanism(s) of the effects of heparin and bFGF remain to be defined, we conclude that they may be a useful adjunct for cardiomyoplasty.

The effect of basic fibroblast growth factor on the blood flow and morphologic features of a latissimus dorsi cardiomyoplasty.

Previous studies designed to determine whether latissimus cardiomyoplasty could be used to revascularize ischemic myocardium showed that after operation the latissimus was ischemic and had severely deteriorated. This study was undertaken to determine whether basic fibroblast growth factor, a potent angiogenic peptide, would improve the vascularity of the latissimus and enhance collateral formation between the muscle of the cardiomyoplasty and ischemic myocardium. In goats, myocardial ischemia was induced with an ameroid constrictor and cardiomyoplasty performed. The latissimus was continuously stimulated electrically at 2 Hz for 6 weeks and given four weekly bolus injections of human recombinant basic fibroblast growth factor (80 micrograms infused into the left subclavian artery). In eight animals, rates of regional blood flow were measured and both the heart and latissimus were evaluated histochemically. The latissimus blood flow rate was 0.114 +/- 0.029 ml/gm per minute, which was three times greater than that of historical controls (chronically stimulated latissimus cardiomyoplasty without basic fibroblast growth factor treatment; 0.042 +/- 0.007 ml/gm per minute, p < 0.05). Associated with the improved blood flow, there was significantly less evidence of skeletal muscle fiber dropout and muscle fibrosis in the animals treated with basic fibroblast growth factor. Latissimus-derived collateral flow to ischemic myocardium developed in five of the eight goats and averaged 0.288 +/- 0.075 ml/gm per minute. This flow was 42.8% +/- 15.7% (n = 5) of the flow required by normal myocardium (which was 0.728 +/- 0.095 ml/gm per minute). This value for latissimus-derived collateral blood flow was almost twice that of the historical controls (24.0% +/- 3.9%), but the increase did not achieve statistical significance (p = 0.08). These results hold the promise that basic fibroblast growth factor treatment might enhance the formation of extramyocardial collaterals to the heart and improve skeletal muscle function.

Basic fibroblast growth factor identified in chronically stimulated cardiomyoplasties.

In the presence of myocardial ischemia, chronic electrical stimulation of a latissimus dorsi (LD) cardiomyoplasty enhances extramyocardial collateral blood flow. We postulated that basic fibroblast growth factor (bFGF) may mediate extramyocardial collateral formation. To test this hypothesis, LDs from goats with cardiomyoplasties were probed for the presence of bFGF by Western blot analysis and immunohistochemistry. Three groups were studied: static LD cardiomyoplasty (group 1); LD cardiomyoplasty stimulated at a 2-Hz frequency for 6 weeks (group 2); and LD cardiomyoplasty electrically stimulated and given human recombinant bFGF (group 3). There was no evidence of bFGF in the left LDs of group 1 by Western blot. Basic fibroblast growth factor-like immunoreactive evidence was found in the left LDs of group 2 goats by both Western blot and immunohistochemistry. In the right LDs of group 2, bFGF-like material was found by immunohistochemistry but not by Western blot, which suggests that the tissue concentrations were low (near the limits of detection). The left LDs of group 3 were positive for bFGF by Western blot and immunohistochemistry. Group 3 right LDs were positive for bFGF by immunohistochemistry. Immunohistochemical findings in group 2 indicate that bFGF is present in goat skeletal muscle. Western blot data from groups 1 and 2 suggest that bFGF may be increased in chronically stimulated cardiomyoplasties. From findings in group 3, we conclude that exogenous bFGF does not downregulate, and may upregulate, endogenous production. These results support the possibility that skeletal muscle bFGF is an important factor in extramyocardial collateral formation.

Acute electrical stimulation increases extramyocardial collateral blood flow after a cardiomyoplasty.

We hypothesized that acute electrical stimulation of a latissimus dorsi cardiomyoplasty would augment the collateral blood flow delivered by the skeletal muscle to the heart. This hypothesis was tested in an animal model (13 goats) of coronary artery disease. Six weeks after a cardiomyoplasty was performed, myocardial collateral blood flow derived from the latissimus dorsi muscle was measured with colored microspheres when the muscle was at rest and during electrical stimulation of the thoracodorsal nerve at 1.25 Hz. The area at risk for ischemia averaged 13.37 +/- 2.08 g (mean +/- standard error), or 18.4% of left ventricular mass (n = 13). At rest, significant skeletal muscle-derived collaterals developed in 9 animals, and formed predominantly to chronic ischemic myocardium (mean +/- standard error, 0.07 +/- 0.02 mL.g-1 x min-1; n = 9), rather than infarct (0.03 +/- 0.02 mL.g-1 x min-1; n = 5), or normal myocardium (0.0005 +/- 0.0001 mL.g-1 x min-1; n = 9). Stimulation increased skeletal muscle-derived collateral blood flow to chronic ischemic areas to 0.38 +/- 0.09 mL.g-1 x min-1 (n = 9) (p < 0.05). During stimulation, the collateral flow was greater in the epicardium (0.46 +/- 0.11 mL.g-1 x min-1) than in endocardium (0.14 +/- 0.09 mL.g-1.min-1) (p < 0.05). This study demonstrates that electrical stimulation of a latissimus dorsi cardiomyoplasty increases extramyocardial collateral blood flow to chronic ischemic myocardium.

Chronic stimulation enhances extramyocardial collateral blood flow after a cardiomyoplasty.

We have previously demonstrated that collateral blood flow can be established between skeletal muscle and myocardium in animals that have undergone a latissimus dorsi cardiomyoplasty. We have also shown that 5 minutes after the thoracodorsal nerve is electrically stimulated at 1.25 Hz, there is a sixfold increase in the collateral blood flow between the latissimus dorsi and the heart. In this experiment, we hypothesized that chronic stimulation of a latissimus dorsi cardiomyoplasty would result in a sustained increase in the latissimus-derived collateral blood flow. In 24 adult male goats, an ameroid constrictor was placed around a branch of the circumflex coronary artery, and a latissimus dorsi cardiomyoplasty was performed. After a rest period of about 1 week, the latissimus dorsi cardiomyoplasties were stimulated continuously at a 2-Hz frequency for 6 weeks. Collateral blood flow between the muscle and the heart was then measured with colored microspheres. Sixteen animals survived to the final experiment, and collaterals developed in 10. In these 10 animals, the latissimus collaterals continuously delivered 0.17 +/- 0.03 mL.g-1 x min-1 (mean +/- the standard error) of blood to ischemic myocardium. This flow represents 24.0% +/- 3.9% of the flow measured to normal myocardium. These results demonstrate that in an animal model of coronary artery disease, chronic electrical stimulation of a latissimus dorsi cardiomyoplasty maintains an elevated level of latissimus-derived collateral blood flow to the myocardium.

Collateral blood flow from skeletal muscle to normal myocardium.

Collateral blood vessels from skeletal muscle to myocardium might supplement intramyocardial collaterals during periods of acute myocardial ischemia. This study was conducted to verify the existence of such collaterals and to measure their contribution to collateral flow. In 12 male goats, the innate coronary collateral system to a moderate size myocardial risk area was defined with colored microspheres, and a latissimus dorsi pedicle flap was then apposed to the heart. After 3 weeks, skeletal muscle to myocardial collaterals were characterized by (a) creation of vascular casts (three animals); (b) estimation of skeletal muscle to myocardial collateral blood flow (three animals); and, (c) measurement of total collateral blood flow to the risk area (innate plus skeletal muscle to myocardial collateral flow). Under a dissecting microscope the vascular casts revealed direct communications from the skeletal muscle which penetrated deeply into the myocardium. With the coronary artery to the risk area open, the estimated myocardial collateral blood flow derived from the muscle flap was 0.01, 0.02, and 0.04 ml/min. With the coronary artery to the risk area closed, there was no significant increase in total coronary collateral blood flow. Although the quantity of blood flow delivered by skeletal muscle collaterals was small, this study demonstrates that clearly identified collateral blood vessels form between skeletal muscle and myocardium in a cardiomyoplasty model. This raises the possibility that, under conditions more favorable to their development, extramyocardial collaterals from skeletal muscle might be exploited to augment the intramyocardial collateral system.

The coronary collateral circulation in normal goats.

This study was undertaken to evaluate the coronary collateral circulation of the goat. Year old, castrated male goats were anesthetized with sodium pentobarbital. A branch of the left circumflex coronary artery was dissected and a snare placed around it. Regional myocardial blood flow was measured by injecting colored microspheres into the left atrium before and during 3 hr of occlusion of this coronary artery. The Area At Risk for infarction, as defined by a left atrial injection of Brilliant Green dye, was divided into a central Ischemic zone and a peripheral Ischemic Border zone. The degree of overlap in the blood flow distributions between the risk and the nonrisk areas was quantitatively assessed by injecting microspheres directly into the artery to the Area At Risk. Baseline blood flow to the normally perfused goat myocardium was 1.13 +/- 0.18 ml/min/g (mean +/- SE). Following occlusion, the flow to the Ischemic zone was 0.07 +/- 0.03 ml/min/g and to the Ischemic Border zone was 0.31 +/- 0.18 ml/min/g. Flow to either zone did not increase during the 3-hr observation period. Overlap at the perimeter of the risk and nonrisk areas was approximately 22%. We conclude that flow to the Ischemic zone is low because the goat has few native collateral blood vessels, and that flow to the Ischemic Border zone is significantly affected by overlap with normal myocardium.

Origin, distribution, and blood flow of bronchial circulation in anesthetized sheep.

This anatomical-physiological study was undertaken to determine the suitability of the sheep for studies of the bronchial circulation. We designated the terminal portion of the bronchoesophageal artery, which runs to the trachea at the carina, as the "carinal" artery. Postmortem injections of india ink (9 sheep) and Batson's solution (4 sheep) into the carinal artery showed that the carinal artery supplied the bronchi of all lobes except the right apical lobe; the mass of the lung containing bronchi perfused represented 88 +/- 1% of total lung mass. Communications were also found between branches of the carinal artery and branches of the systemic arteries supplying the visceral pleura. In three of six sheep, the carinal artery wedge pressure measured in vivo indicated that patent collaterals were present, but that their incidence is variable. In additional postmortem casts of the aorta made in four sheep after ligation of the carinal artery, the bronchial microcirculation was not entered by the perfusing medium. These data indicate that the collaterals do not contribute significantly to bronchial blood flow and that the carinal artery is the major source of bronchial blood flow in the sheep. In 19 sheep, carinal artery flow, measured electromagnetically, was 0.46 +2- 0.09 ml . min-1 . kg body wt-1. Bronchial blood flow normalized for the weight of the lobes in which bronchi perfused by the carinal artery were located was 3.97 +/- 0.48 ml . min-1 . 100 g lung-1 (n = 11); carinal artery blood flow was 0.39 +/- 0.03% of cardiac output (n = 5). During the 90 min of observation, at normal aortic blood pressure (103 +/- 3.4 Torr), carinal artery blood flow was stable. In conclusion, determination of carinal artery blood flow affords a reliable approach to the bronchial circulation in the sheep.