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The morphological effects on human endothelial cells of phorbol 12-myristate 13-acetate (PMA) and of agents that increase intracellular cAMP concentration were studied. The adenylate cyclase activator forskolin (10 IAM), the cyclic nucleotide phosphodiesterase inhibitor methylisobutylxanthine (100 ,uM), dibutyryl-cAMP (10 IAM), histamine (10 IAM), and PMA (0.1 ,LM) significantly altered the morphology of human aortic and umbilical vein endothelial cells in primary cultures. These effects reached a maximum 40-80 min after the effector addition and became negligible 30-60 min after its removal. PMA and forskolin were strongly synergistic in altering endothelial cell morphology. All the effects of cAMPelevating compounds and ofPMA were abolished completely by 1 ,uM colchicine. In explants taken from human adult or child aortas, forskolin and PMA produced alterations in endothelial morphology qualitatively identical to those observed in endothelial cell cultures. Endothelium in these preparations closely resembled that found in zones of expected altered hemodynamic stresses of human aorta. Our data suggest that the morphology of endothelium in vivo may be regulated by separate or synergistic action of hormone-dependent adenylate cyclase and of inositol phospholipid turnover systems and might be important for maintenance of endothelial monolayer integrity under normal physiological and pathological conditions. MATERIALS AND METHODSMedium 199 (Earle salts), fetal calf serum, Dulbecco's phosphate-buffered saline (PBS), L-glutamine, penicillin, streptomycin, and sodium pyruvate were from GIBCO. Dispase (neutral protease from Bacillus polymixa) was from Boehringer Mannheim. Phorbol 12-myristate 13-acetate (PMA), 4a-phorbol 12,13-didecanoate, histamine, 1-methyl-3-isobutylxanthine, N6,02-dibutyryl-cAMP (Bt2cAMP), colchicine, bovine serum albumin (BSA, essentially fatty acid free) were supplied by Sigma. Forskolin was from Calbiochem. Drugs were dissolved in dimethylsulfoxide or ethanol (Merck) and stored as stock solutions in aliquots at -80°C. Prior to use the aliquots were diluted with medium 199 and 0.5% BSA or with growth medium [medium 199 supplemented with 25 mM Hepes, 2 mM L-glutamine, 1 mM pyruvate, penicillin at 100 units/ml, streptomycin at 100 ,ug/ml, 10% (vol/vol) fetal calf serum] and sterilized by filtration through 0.22-,um filters (Nalgene).Primary cultures of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were obtained as described (15,16), with minor modificationdispase was used instead of collagenase. Seeding density was 5-8 X 104 cells per cm2. Only confluent (7-9 days after seeding) cultures were used for experiments. The main characteristics of these cultures have been reported (16). For morphometry, cultures were stained with silver nitrate (17) and photographed. Cell shape was analyzed using a MOP-3 digitizer (Reichert, Jung, Vienna) and described in terms of shape index (SI), where SI = 4r x area/(perimeter)2. All the experiments were performe...

Scanning electron microscope studies of the aorta and other major arteries have been performed in alloxan-induced diabetic rabbits. After 5 weeks, a variety of structural abnormalities of the endothelial lining were detected including a significant increase in the number of argyrophilic cells and an increased number of craters or openings in the endothelial junctional region. Evidence of more extensive micro-damage was present after 5 months duration of diabetes. These zones with structural changes in the endothelial lining of major vessels seem to be areas of high predilection to atherosclerosis in diabetes.

Immunomorphological staining of specimens prepared from human carotid arteries with anti-collagen type I antibodies reveals large amounts of type I collagen in the subendothelium of lipid fibrous plaques. Collagen type Icontaining structures, once in direct contact with blood after plaque rupture, can serve as potential targets for selective delivery of liposomes and erythrocytes to these areas. To verify this rationale, ['4C]cholesterol oleate-containing liposomes were conjugated with bovine or human anti-collagen type I antibodies or human plasma fibronectin. Biotin derivatives of human anti-collagen type I antibody were coupled to human erythrocytes. Modified liposomes and erythrocytes were perfused in situ through segments of bovine, rabbit, or human arteries partially denuded with a balloon catheter prior to perfusion. After perfusion, the control and denuded areas were excised and subjected to scanning electron microscopic analysis and measurements of associated radioactivity. It was found that conjugates of liposomes or erythrocytes with anti-collagen type I antibodies or fibronectin are selectively bound by endothelium-free zones of arterial segments. Carrier-directed targeting of drug-laden liposomes and erythrocytes to thrombosis-prone areas of arterial lumen is discussed.