Michael G. Magno

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 (·O2−) was measured at 2 weeks after surgery. SVGs demonstrated increased ·O2− 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 ·O2−, 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 125I-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.

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.

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.

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.

Myofibroblast Involvement in Glycosaminoglycan Synthesis and Lipid Retention during Coronary Repair

Myofibroblasts of adventitial origin have been linked to neointimal formation and remodeling after coronary injury. Accordingly, the goal of this study was to examine whether myofibroblasts contribute to focal accumulation of glycosaminoglycans (GAGs) and lipids during coronary repair. GAG synthesis was assessed by ex vivo labeling of balloon-injured porcine coronary arteries with 14C-glucosamine. The synthesis of total GAGs transiently increased at 8 days in the normolipemic model (a 2.2-fold increase over baseline, p < 0.05). The majority of newly synthesized GAGs were sensitive to chondroitin ABC lyase (chondroitin/dermatan sulfate GAGs). Versican was localized to myofibroblast-rich regions in the adventitia and neointima [positive for α-smooth muscle (SM) actin, negative for h-caldesmon and SM myosin heavy chain]. In contrast, the adjacent SM-rich media showed no increase in versican expression. The association between injury-induced GAG accumulation and lipid retention was examined at 2 weeks after coronary injury in the hyperlipemic model. Lipid (Oil Red O) accumulated in the neointima and adventitia, but not in the adjacent media. Coronary repair under hyperlipemic conditions was associated with macrophage infiltration (19 ± 5 vs. 3 ± 2% of neointimal cells in normolipemic animals, p < 0.001) and increased neointimal formation (1.8 ± 0.5 vs. 1.0 ± 0.3 mm2 in normolipemic animals, p < 0.01). In conclusion, this study demonstrated a transient increase in GAG synthesis following coronary injury. Chondroitin sulfate proteoglycans (e.g., versican) were rapidly synthesized by activated adventitial and neointimal cells which could contribute to early lipid retention in injured vessels.

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.

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.

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.

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.

Techniques to enhance extramyocardial collateral blood flow after a cardiomyoplasty.

ObjectiveChronic stimulation of a cardiomyoplasty was combined with low-dose infusion of heparin into the arterial supply of the cardiomyoplasty in order to determine if latissimus-derived collateral blood flow could be further enhanced. Summary Background DataAcute and chronic stimulation of a latissimus dorsi cardiomyoplasty increased extramyocardial collateral blood flow to 35 ± 9% and 27 ± 5%, respectively, of normal myocardial blood flow. MethodsA model of coronary artery disease was created with an ameroid constrictor in goats, and a cardiomyoplasty was performed. Heparin (15 to 50 U/h) was delivered into the left subclavian artery for a period of 4 weeks. Simultaneously, the latissimus dorsi was chronically stimulated at 2 Hz. ResultsChronic ischemic myocardium received a collateral flow per gram from the skeletal muscle equivalent to 11.8 ± 5.2% of the blood flow to normal myocardium. The extramyocardial collateral flow correlated with the latissimus muscle flow (r = 0.72). ConclusionsEnhancement of extramyocardial collateral flow was not found with heparin treatment. In view of the correlation of extra-coronary collateral flow with latissimus muscle flow, the lack of a heparin effect may have been due to low latissimus blood flow. These results suggest that extramyocardial collateral blood flow to the myocardium is highest if the blood flow to the latissimus dorsi muscle is maintained.