John Dillman

Clonal markers in the study of the origin and growth of human atherosclerotic lesions.

The X-linked enzyme, glucose-6-phosphate dehydrogenase (G-6-PD) was used as a cellular marker to study the clonal characteristics of human atherosclerotic lesions from females heterozygous for G-6-PD isoenzymes. Portions of uninvolved aortic wall contained both isoenzyme types (A and B), and their isoehzyme patterns were used to establish criteria for polyclonal lesions. Portions of uterine leiomyomas contained predominantly one isoenzyme type (either all A or all B) and their isoenzyme patterns were used to establish criteria for monoclonal lesions. These techniques were used to address three questions concerning atherogenesis. First, evidence for the monoclonal origin of fibrous-capped plaques was provided by the findings that small plaques had G-6-PD isoenzyme distributions similar to those of leiomyomas; that in large plaques with multiple portions assayed for G-6-PD, a large proportion (25 of 26, 96%) of plaques had monoclonal characteristics; and that multiple monoclonal portions were present in the same plaque. Second, the role of the fatty streak as a precursor of fibrous plaques was supported by the demonstration that a proportion (11 of 66, 16.7%) of fatty streaks contained isoenzyme patterns intermediate between those of polyclonal uninvolved aortic wall and monoclonal leiomyomas. Increased cellularity of fatty streaks correlated with increased deviation of isoenzyme pattern toward monoclonality. Third, the assay of portions of both small and large plaques provided no evidence for clonal selection as plaques increase in size.

MONOCLONAL CHARACTERISTICS OF ORGANISING ARTERIAL THROMBI: SIGNIFICANCE IN THE ORIGIN AND GROWTH OF HUMAN ATHEROSCLEROTIC PLAQUES

The clonal characteristics of 26 arterial thrombi at different stages of organisation were determined using the X-linked enzyme, glucose-6-phosphate dehydrogenase (G.-6-P.D.), as a clonal marker in 13 women heterozygous for electrophoretically separable G.-6-P.D. isoenzymes. A gradation of increasing monoclonality was observed with increasing organisation of the thrombi, such that only 21% of poorly organised (red) thrombi displayed monoclonal characteristics similar to those of atherosclerotic plaques, whereas 78% of moderately organised (pink) thrombi and 91% of well-organised (white) thrombi showed such characteristics. These results provide objective evidence for the role of thrombosis in the formation of human atherosclerotic plaques.

Thrombolysis with recombinant tissue plasminogen activator in atherosclerotic thrombotic occlusion.

Human tissue plasminogen activator holds promise for the dissolution of coronary thrombi by intravenous administration and without systemic anticoagulation. Prior animal experiments have been conducted only in vessels without disease. To test the thrombolytic efficacy of recombinant tissue plasminogen activator in the presence of diseased intima, an established model of atherosclerosis was utilized. The aorta of 16 New Zealand white rabbits (2 to 3 kg) was made atherosclerotic by balloon endothelial denudation and concurrent 1% cholesterol feeding for 8 weeks. An aged (24 hour) heterologous (human) clot, labeled with I-125 fibrinogen was injected into the distal aorta and produced thrombotic occlusion. After 1 hour of thrombosis (control period), recombinant tissue plasminogen activator (100,000 IU approximately equal to 1 mg protein, n = 6) or streptokinase (100,000 IU, n = 5) or saline solution (n = 5) was systemically infused over 30 minutes. Serial blood samples, obtained to determine fractional change in blood radioactivity over time, showed a fourfold increase of blood radioactivity after tissue plasminogen activator and streptokinase infusion compared with the control period (47,400 +/- 3,300 [mean +/- standard error] versus 11,800 +/- 300 counts/min, p less than 0.001). Time to 50% of maximal thrombolysis was 41 +/- 14 minutes (+/- standard deviation) for tissue plasminogen activator versus 63 +/- 16 minutes for streptokinase (p less than 0.01). In six of six rabbits receiving tissue plasminogen activator and four of five rabbits receiving streptokinase, reestablishment of distal aortic flow was detected via the indwelling catheter within 25 minutes of drug infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Atherosclerosis in the hypercholesterolemic hare: Comparison of coronary artery lesions induced by dietary cholesterol in the hare and the rabbit

Atherosclerotic lesions in coronary arteries were compared in 10 hybrid hares and 14 rabbits after induction of hypercholesterolemia, using a cholesterol-enriched diet. All proximal portions of hare coronary arteries contained intimal lesions, often with severe luminal stenosis. These lesions were characterized by the presence of foam cells, smooth muscle cells, and areas of atheronecrosis. Foam cells were also found focally in the media. As part of the intimal changes, iron deposition was present in 65% and calcification was present in 32.5% of proximal segments examined. The proportion of segments with intimal lesions and the intima/media cross-sectional area ratios (I/M ratios) were greatest in proximal segments with stepwise decreases in the mid and distal segments. As area of myocardial infarction was present in one hare. In contrast, 46.5% of proximal segments of rabbit coronary arteries had no intimal lesions and those lesions present had no calcium or iron deposition. No infarction was observed in rabbit hearts. The proportion of segments with lesions and the mean I/M ratios were significantly greater in the hare than the rabbit, with proximal and mid coronary segments showing the most marked differences. The hare appears to develop coronary artery lesions more like those seen in man, with high grade, proximal stenoses occurring uniformly in hypercholesterolemic animals. In contrast, the atherosclerosis developing in rabbit coronary arteries is less uniform and includes involvement of intramyocardial arterioles. The hare offers several advantages as a model of human atherosclerosis.

Cholesterol-induced atherosclerosis. Clonal characteristics of arterial lesions in the hybrid hare.

Utilizing the observation that a majority of human atherosclerotic fibrous plaques show monoclonal characteristics, we carried out this study to determine the clonal characteristics of cholesterol-induced atherosclerosis in the hybrid hare. If this is a valid model for human atherosclerosis, the lesions produced in the aorta should be monoclonal. Glucose-6-phosphate dehydrogenase (G-6-PD) was used as an X-linked cellular marker in the female hybrid hare (Lepus timidus × Lepus europaeus), which is heterozygous for electrophoretically separable isoenzymes of G-6-PD. Hares were fed cholesterol over either a 6-month ora l 6-month period, and the easily dissectable lesions in the aorta and common iliac arteries were assayed for isoenzyme activity at these times. Of the 93 lesions assayed, all had polyclonal characteristics except a single monoclonal lesion found in an animal fed cholesterol over a 16-month period. Hares fed over the 16-month period showed lesions with isoenzyme patterns having a significantly higher contribution of L. timidus isoenzyme than those found in underlying media. This suggested that a selection of cells with the L. timidus X-chromosome had taken place, but the degree of this selection was not great enough to allow any of the lesions to be defined as monoclonal.

Dipyridamole and Nifedipine—Studies on Their Protective Effects for Heart Graft Arterial Stenosis:

The etiology of accelerated graft coronary arterial stenosis (CAS) was stud ied in a rat heterotopic heart transplantation model. Spontaneously hyperten sive rats (SHR), Wistar rats (WR), and Lewis rats (LEW) were used. Thirty-four recipients rats fed a hypercholesterol diet (HCD) and saline for eight weeks were divided into six groups. Group 1 and 2 (5 rats each) were hypertensive isograft models (SHR to SHR), group 2 being treated with cyclos porine (CsA: 10mg/kg/day orally). The other 4 groups (6 rats each) represented the allograft models. Group 3 (LEW to WR) was the CsA-treated normotensive model, and Groups 4, 5, and 6 (LEW to SHR) were CsA-treated hypertensive models. Additionally, group 5 was treated by dipyridamole (DP; 25mg/kg/day orally) and group 6 was by nifedipine (NF; 3mg/kg/day orally). The coronary stenosis index (CSI) was calculated in each case and the proportion of arteries affected (AR, %) was estimated in each group. AR and CSI were 6.1% and 0.53 ± 0.76 in group 1, 3.6 % and 0.50 ± 0.71 in group 2, 23.7 % and 1.85±0.52 in group 3, 58.2 % and 2.45 ±0.18 in group 4, 32.3% and 2.25 ±0.29 in group 5, and 27.7% and 1.93 ± 0.48 in group 6. AR and CSI were significantly increased in group 3 vs group 1 (p < 0.05) and in group 4 vs group 3 (p < 0.05). AR was reduced in groups 5 and 6 vs group 4 (p < 0.05), and CSI was also reduced in group 6 vs group 4(p < 0.05). In conclusion, hypercholesterolemia and hyper tension were recognized as risk factors for CAS aggravation in allografts but failed to affect the coronary arteries of isografts. Both antiatherogenic agents reduced the number of affected vessels, but only NF reduced the degree of CAS. However, neither controlled graft CAS completely.