Donald C. MacLeod

Proliferation and extracellular matrix synthesis of smooth muscle cells cultured from human coronary atherosclerotic and restenotic lesions

OBJECTIVES The purpose of this study was to examine the proliferative capacity and extracellular matrix synthesis of human coronary plaque cells in vitro. BACKGROUND Common to both primary atherosclerosis and restenosis are vascular smooth muscle cell proliferation and production of extracellular matrix proteins. The applicability to humans of experimental animal models of these processes has been questioned. METHODS Primary atherosclerotic and restenotic lesions were excised by percutaneous directional coronary atherectomy in 93 patients. Smooth muscle cells were cultivated by an explant technique and identified by their morphology in culture, ultrastructural features under electron microscopy and immunostaining using monoclonal antibodies to smooth muscle cell alpha-actin. Proliferation in secondary culture was assessed with growth curves and the synthesis of collagen and sulfated glycosaminoglycans by the incorporation of 3H-proline and 35S-sulfate, respectively. These studies were also performed in cells derived from human umbilical artery media. RESULTS Success rates for primary (45%) and secondary (12%) culture of coronary cells were not influenced by clinical variables or lesion category. Primary culture success was improved by the presence of organized thrombus in the plaque and in relation to increased maximal cell density of the atherectomy specimen. Restenotic cells displayed more rapid growth than did cells of primary atherosclerotic origin, which grew in a manner similar to that of umbilical artery cells. Mean calculated population-doubling times for the three cell groups were 52 h (95% confidence interval [CI] 48 to 58 h), 71 h (95% CI 62 to 83 h) and 74 h (95% CI 65 to 84 h), respectively. Restenotic and primary atherosclerotic cells did not differ in the synthesis of collagen ([mean +/- SEM] 0.034 +/- 0.004 vs. 0.033 +/- 0.004 nmol isotope.microgram protein-1, p = NS) or sulfated glycosaminoglycans (11.47 +/- 1.07 vs. 15.37 +/- 3.10 nmol isotope.microgram protein-1, p = NS), but the coronary cells synthesized significantly more collagen and sulfated glycosaminoglycans than did umbilical artery cells (0.019 +/- 0.004 and 5.43 +/- 1.00 nmol isotope.microgram protein-1, respectively, both p < 0.05). CONCLUSIONS These data indicate that increased smooth muscle cell proliferation contributes to coronary restenosis in humans and support the concept that the extracellular matrix synthesis of adult smooth muscle cells is important to lesion formation.

Predictive value of reactive hyperemic response on reperfusion on recovery of regional myocardial function after coronary angioplasty in acute myocardial infarction.

BACKGROUND The objective of the study was to determine the coronary vasodilatory reserve in reperfused myocardium in patients with acute myocardial infarction and its relation to regional myocardial function. METHODS AND RESULTS The study population consisted of 22 patients with acute myocardial infarction who underwent successful coronary angioplasty. The vasodilatory reserve in the reperfused myocardium was assessed quantitatively using computer-assisted digital subtraction cine-angiography immediately after angioplasty and at follow-up angiography before hospital discharge. Myocardial contrast medium appearance time and density were determined before and after pharmacological hyperemia induced by an intracoronary injection of 12.5 mg papaverine. Global and regional left ventricular functions were determined from contrast angiography. After papaverine, the mean contrast medium appearance time decreased significantly from 3.5 +/- 0.7 to 2.7 +/- 0.7 cardiac cycles (P < .000005) immediately after successful coronary angioplasty and from 3.8 +/- 0.7 to 2.7 +/- 0.9 cardiac cycles (P < .000005) at angiography before hospital discharge. The mean contrast medium density increased significantly from 48.7 +/- 13.8 to 61.0 +/- 19.0 pixels (P < .003) and from 49.6 +/- 19.7 to 80.3 +/- 29.6 pixels (P < .000005), respectively. As a consequence, the calculated coronary flow reserve increased significantly from 1.8 +/- 0.7 to 2.6 +/- 1.0 (P < .0008). The global ejection fraction increased significantly from 52 +/- 12% to 58 +/- 14% (P < .03), primarily because of a significant improvement in the regional myocardial function of the infarct zone from 20.8 +/- 9.0% to 26.0 +/- 10.5% (P < .001). Coronary flow reserve correlated well with regional myocardial function both during the acute phase (R = .79, P < .002) and at follow-up angiography (R = .82, P < .000004). Interestingly, coronary flow reserve measurement on reperfusion, immediately after angioplasty, correlated significantly with regional myocardial function at follow-up angiography (R = .81, P < .00003). CONCLUSIONS The results indicate that there is a pharmacologically inducible vasodilatory reserve in reperfused ischemic myocardium after successful coronary angioplasty in patients with acute myocardial infarction and that this is increased at 10-day follow-up angiography. More important, the degree of reactive hyperemic response on reperfusion has a predictive value regarding the ultimate degree of recovery of regional myocardial function. Quantitative assessment of reperfusion may be useful in investigating the role of coronary reperfusion and salvage of myocardial function.

Histologic characteristics of tissue excised during directional coronary atherectomy in stable and unstable angina pectoris

Unstable angina is an acute coronary syndrome associated with substantial short- and medium-term morbidity and mortality.1 The understanding of the pathogenesis of this syndrome has been based largely on postmortem studies of coronary arteries2 and supported by indirect evidence of coronary thrombosis in relation to the syndrome.3–5 Because directional coronary atherectomy is unique in extracting intact atheromatous tissue during coronary recanalization, it may facilitate the study of the processes taking place in the vessel in different coronary syndromes. In the present study the histopathologic characteristics of atherectomy samples retrieved in 93 patients with stable or unstable angina pectoris were compared and related to different clinical variables.

Nicorandil and cardiovascular performance in patients with coronary artery disease

To establish the cardiovascular profile of nicorandil in patients with coronary artery disease, we recently conducted three studies at our institution. In two groups of patients undergoing cardiac catheterization, the effects of 20 mg nicorandil sublingually (s.l.) on, first, left ventricular hemodynamics (n = 10) and, second, coronary vasodilatation (n = 11) were investigated. In the first group, despite a significant decrease of 12% in left ventricular systolic pressure, heart rate did not increase significantly after nicorandil. Both left ventricular end-dia-stolic pressure (−43%) and the time constant of early isovolumic relaxation (−11%) decreased, whereas peak VCe and Vmax increased (+19%) (all significantly). In the second group, as mean aortic pressure decreased (−13%, p < 0.05), coronary sinus blood flow did not change significantly, and calculated coronary vascular resistance tended to decrease (−10%). Myocardial oxygen consumption decreased significantly by 14%. Quantitative coronary angiography confirmed a significant increase in the mean diameter of nonstenotic coronary artery segments (+14%, n = 43) and, importantly, in mean obstruction diameter of stenotic segments (+14%, n = 7) after s.l. nicorandil. In a third continuing study, the effects of intracoronary (i.e.) nicorandil (6 μg/kg) and isosorbide dinitrate (2 mg) on the epicardial coronary arteries were investigated in 10 patients undergoing coronary angioplasty. In nonstenotic coronary artery segments, mean coronary diameter increased significantly after either nicorandil (+ 12%) or isosorbide dinitrate (+ 17%). In stenotic segments, however, where the increase in obstruction diameter (+ 20%) after i.e. nicorandil was significant, the 8% increase of isosorbide dinitrate was not. Importantly, there was a significant additional increase of 13% in obstruction diameter when nicorandil was administered after isosorbide dinitrate, but no such additional effect was observed when isosorbide dinitrate was administered after nicorandil. Nicorandil s.1. in a dose of 20 mg reduces both preload and afterload but is free of cardiac depressant effects. It decreases myocardial oxygen demand but maintains coronary sinus blood flow and induces vasodilatation in both nonstenotic and stenotic epicardial coronary arteries. Both i.e. nicorandil and isosorbide dinitrate induce significant vasodilatation of nonstenotic coronary segments, but nicorandil appears to be more potent than isosorbide dinitrate in dilating stenotic coronary segments.

Acute and long-term outcome of directional coronary atherectomy for stable and unstable angina

The clinical efficacy and safety of directional coronary atherectomy for the treatment of stable and unstable angina were assessed in 82 patients with stable and 68 patients with unstable angina. Therefore, clinical and angiographic follow-up was obtained in a prospectively collected consecutive series of 150 atherectomy procedures. Restenosis was assessed clinically and by quantitative angiography. The overall clinical success rate of atherectomy for patients with unstable and stable angina was 88% and 91%, respectively. No significant differences were found for in-hospital event rates between the unstable and stable angina groups: death (1.5% vs 0%), myocardial infarction (10% vs 6%), and emergency bypass operation (3% vs 2%). These clinical events were related to the occurrence of abrupt occlusions (8.8% in patients with stable and 6.1% in those with unstable angina; p = NS). Clinical follow-up was achieved in 100% of the patients with stable and unstable angina at a mean interval of 923 and 903 days, respectively. Two-year survival rates were 96% and 97% in the populations with unstable and stable angina, respectively. There were no significant differences with respect to bypass surgery and angioplasty, but event-free survival at 2 years was significantly lower in the unstable (54%) than the stable (69%) angina group. Quantitative coronary angiography did not detect any difference in luminal renarrowing during the 6-month angiographic follow-up period. Although directional coronary atherectomy can be performed effectively in patients with unstable and stable angina, the long-term clinical outcome was less favorable in the unstable angina group.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and histological determinants of smooth-muscle cell outgrowth in cultured atherectomy specimens: importance of thrombus organization.

Background:Coronary atherectomy provides a unique opportunity to obtain plaque tissue from a wide variety of clinical syndromes. We investigated the relation between the clinical status and histopathological substrate of tissue retrieved during directional coronary atherectomy and the proliferative and migratory potential of smooth-muscle cells judged from successful outgrowth during cell culture. Methods:After directional coronary atherectomy, tissue samples were examined macroscopically, divided into two equal pieces, and separately subjected to cell culture and histopathological study. Cell culture was performed using an explant technique. In-vitro smooth-muscle cell outgrowth was related to clinical and histological variables. Results:Atherosclerotic tissue was obtained from 98 consecutive atherectomy procedures. Histological examination revealed a broad spectrum of appearances, ranging from complex atheroma containing dense fibrous tissue, calcium deposits, macrophages, and necrotic debris to neointimal proliferation and organized thrombi. Smooth-muscle cell outgrowth was observed in 43 of the 98 samples (44%). Although not affected by any of the clinical variables, cell outgrowth was influenced by histological variables, in particular the presence of organizing thrombi. Outgrowth was successful in eight out of 10 samples with thrombus (80%) and in only 35 out of 88 (40%) without (P=0.03). Conclusion:The presence of organizing thrombi in the retrieved tissue facilitates smooth-muscle cell outgrowth and suggests an enhanced proliferative and migratory potential. These findings may be relevant to the understanding of neointimal proliferation in coronary syndromes where mural thrombosis is likely to occur.

Effect of acadesine on myocardial ischaemia in patients with coronary artery disease

Acadesine, an adenosine regulating agent, attenuates the adverse effects of ischaemia on ventricular function in animals. This study examined its influence on pacing-induced ischaemia in 47 patients undergoing coronary angiography. After 15 min of recovery from control pacing, an infusion of acadesine (5, 10, 20, 50 mg/kg i.v.) was commenced and after a further 15 min the protocol was repeated with the infusion continued. At higher doses, minor beneficial effects on ejection fraction and myocardial lactate metabolism were observed. Haemodynamics were unaffected. Systemic lactate rose in relation to acadesine, up to 60% (P < 0.001 versus placebo). The data may indicate that acadesine stimulates anaerobic glycolysis in man.

Increased extracellular matrix synthesis by smooth-muscle cells obtained from in vivo restenotic lesions by directional coronary atherectomy

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Coronary Vasodilatory Action of Elgodipine in Coronary Artery Disease

The effects of intravenous elgodipine, a new second-generation dihydropyridine calcium antagonist, on hemodynamics and coronary artery diameter were investigated in 15 patients undergoing cardiac catheterization for suspected coronary artery disease. Despite a significant decrease in systemic blood pressure, elgodipine infused at a rate of 1.5 micrograms/kg/min over a period of 10 minutes did not affect heart rate and left ventricular end-diastolic pressure. The contractile responses during isovolumic contraction showed a slight but significant increase in maximum velocity (56 +/- 10 to 60 +/- 10 seconds-1; p less than 0.005), whereas the time constant of early relaxation was shortened from 49 +/- 11 to 44 +/- 9 ms (p less than 0.05). Coronary sinus and great cardiac vein flow increased significantly by 15 and 26%, respectively. As mean aortic pressure decreased, a significant decrease in coronary sinus (-27%) and great cardiac vein (-28%) resistance was observed, while the calculated myocardial oxygen consumption remained unchanged. In all, 69 coronary segments (including 13 stenotic segments) were analyzed quantitatively using computer-assisted quantitative coronary angiography. A significant increase in mean coronary artery diameter (2.27 +/- 0.53 to 2.48 +/- 0.53 mm; p less than 0.000001), as well as in obstruction diameter, (1.08 +/- 0.29 to 1.36 +/- 0.32 mm; p less than 0.02), was observed. The results demonstrate that elgodipine, in the route and dose described, induces significant vasodilatation of both coronary resistance and epicardial conductance vessels, without adverse effects on heart rate, myocardial oxygen demand and contractile indexes.

Analysis of VNTR loci amplified by the polymerase chain reaction for investigating the origin of intimal smooth muscle cells in a coronary artery lesion developing after heart transplantation in man

Focal intimal thickening is a feature of primary atherosclerotic coronary lesions and restenotic lesions following percutaneous transluminal coronary angioplasty and other forms of vascular intervention, where it is the nonspecific response of the vessel wall to injury. The principal cellular component of the coronary plaque is the smooth muscle cell.’ Whether the smooth muscle cell in human coronary lesions is derived from cells circulating in the blood or from the vessel wall itself remains a matter for debate. On the basis of animal studies, it is generally presumed that the tunica media or subintimal space is the source of intimal smooth muscle cells.2-5 However, there is sound experimental evidence that smooth muscle cells recognized in a vascular plaque may originate in mural thrombus and not in the adjacent vessel wall. 6-8 Regardless of their source, these cells would normally be indistinguishable. Atherosclerotic lesions developing in the coronary arteries after orthotopic heart transplantation provide a unique opportunity to pursue the origin of cells within the coronary atherosclerotic plaque, as the donor and recipient invariably differ in genotype. Genetic differences may be demonstrated by the electrophoretic analysis of alleles from the highly polymorphic variable number of tandem repeats (VNTR) gene loci that occur widely in the human genome.g,10 The Dl7S5/Dl7S30 VNTR locus, the DlS80/ DlS58 VNTR locus, and the Apo B 3’ VNTR locus (the hypervariable 3’ region of the apolipoprotein B gene) represent independent, highly polymorphic deoxyribonucleic