Dialyzed serum from clotted monkey blood ("blood serum") promotes the proliferation of monkey arterial smooth muscle cells in culture, but dialyzed serum prepared from recalcified platelet-poor plasma ("plasma serum") is much less effective. Addition of platelets and calcium to platelet-poor plasma increases the activity of plasma serum to the same level achieved with blood serum. Furthermore, addition to plasma serum of a platelet-free supernatant prepared by exposing purified platelets to thrombin also stimulates the proliferation of smooth muscle cells. Thus, much of the growth-promoting activity of dialyzed serum is directly or indirectly derived from platelets. This finding has important implications for the response of arteries to localized injury and provides a key to further understanding of the role of factors derived from blood serum in promoting cell proliferation in vitro.We have been studying the growth of arterial smooth muscle cells (SMC) in culture as part of an attempt to determine why these cells accumulate focally in atherosclerosis (1). Since our working hypothesis has been that atherosclerosis is an exacerbated arterial response to local endothelial injury, we have been interested in the fact that SMC, like most other diploid, nontransformed cells, do not proliferate in culture except in the presence of blood serum. We have been attempting to identify the serum factors involved, in order ultimately to test the possibility that endothelial injury increases their concentration in the subendothelial space and thereby promotes SMC proliferation in the intima. We have already found (1, 2) that serum lipoproteins are necessary for optimal cell growth in culture, and now wish to report studies of a nondialyzable serum component that is probably not a lipoprotein and that appears to be derived from platelets. MATERIALS AND METHODS Procedures described in detail elsewhere (1-3), were used to subculture SMC from explants of the intima and inner media of the thoracic aorta of pigtail monkeys (Macaca nemestrina). The explants were grown in a modified Dulbecco-Vogt medium (3) based on that originally described by Eagle, and containing 5% or 10% pooled blood serum from the same species of monkey. SMC from the explants were subcultured in a similar medium that contained 5% blood serum and lacked streptomycin. Cells from the third to sixth subculture were used to test the effect of serum factors upon cell proliferation. The assay procedure used was similar to that of Paul et al. (4) equal numbers in 35-mm Falcon plastic petri dishes. The reproducibility of cell plating was confirmed in each experiment. The medium was changed three times per week. For the first 7 days the medium .contained 1% 'pooled monkey blood serum. Thereafter, the 1% serum was replaced with the different serum fractions to be tested. Each fraction was added in an amount that corresponded to that provided by 5% monkey blood serum, and subsequent cell proliferation was compared with that obtained in medium containing 5% blood ser...

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Morphologic studies resulting from events that occur during the development of the lesions of atherosclerosis were studied In chronic, diet-induced hypercholesterolemia In a series of nonhuman primates. Within 12 days of hypercholesterolemia In Macaca nemestrina, monocytes became adherent to the surface of the endothelium. These monocytes appeared to migrate subendothelially, accumulate lipid, and become lipld-laden macrophages (foam cells). Within a month, a "seroflbrinous insudate" formed together with variable numbers of subendothellal llpid-laden macrophages. By the second month, foam cells increased In number, often in multilayers, to form a fatty streak. Concomitantly, the luminal surface of the arteries became increasingly Irregular due to the subendothelial accumulation of foam cells. Numerous monocytes continued to attach to the endothelial surface over the fatty streaks, and many of them appeared to enter the Intlma and participate In the growth of the fatty streaks. Lipid-laden smooth muscle cells appeared in small numbers and formed two to four layers between the macrophages and the internal elastic lamella at 2 to 3 months. During the third month of hypercholesterolemla, endothelial cell continuity over the llpid-laden macrophages became Interrupted, exposing the underlying foam cells to circulating blood. Foam cells were then readily observed In whole blood smears, suggesting that many of the lipld-laden macrophages leave the intima and enter the circulation.After 4 months, significant endothelial denudation was found In the lilac artery and many exposed macrophages were covered by adherent platelets in the form of a mural thrombus. Thus, the early components of atherosclerosis induced by chronic hypercholesterolemla centered around the monocyte-macrophage and its interaction with endothelium In the induction of the fatty streak. Subsequent changes that lead to macrophage-smooth muscle Interactions, platelet-macrophage Interactions, and platelet-endothelial interactions appeared to set the stage for the development of more advanced proliferatlve lesions. (Arteriosclerosis 4:323-340, July/August 1984)

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Bleeding after CPB has been difficult to characterize and its treatment equally difficult to standardize. The complexity of this problem is related to the hemostatic process, the technical variations in the operative procedures, and the many uncontrolled variables associated with CPB, including the effects of anesthetic or pharmacologic agents, the nature of the priming solution, hemodilution, hypothermia, the type of oxygenator, and the use of transfused blood products. Although there are multiple and generally predictable complex changes in the hemostatic mechanism during CPB, the temporary loss of platelet function is the most common and clinically relevant. This transient platelet dysfunction occurs in all patients undergoing CPB; however, it only causes excessive bleeding in a small percentage of patients. Unfortunately, it has not yet been possible to predict which patients will develop hemorrhagic complications, although prolonged pump times are a contributing risk factor. Over the past decade there has been extensive investigation into the management of bleeding associated with CPB, provoked primarily by the increased awareness of transfusion- transmitted viral diseases and the inappropriately excessive use of homologous blood products. Several approaches to autotransfusion of shed blood and autologus blood donation have been developed to minimize perioperative homologous blood transfusion. Pharmacologic agents such as desmopressin, aprotinin, and topical fibrin glues have also been introduced to improve hemostasis during CPB. The protease inhibitor aprotinin is particularly promising in the reduction of bleeding associated with CPB when given prophylactically. Aprotinin may provide new insights into the mechanism of CPB-induced platelet dysfunction. Desmopressin is indicated only for the treatment of bleeding after CPB. The management of bleeding associated with CPB will undoubtedly

These studies provide new insight into the complex mechanisms wherby hyperlipidemia causes progressive atherosclerosis. It has been shown that physical injury to the endothelial lining of arteries sets off a process which probably is an attempt at healing the injury but which can lead to atherosclerosis. It has also been found that chemical agents such as homocystine can produce a similar series of events leading to atherosclerosis. These events include focal loss of endothelium, exposure of subendothelial connective tissue, and adherence of platelets followed by release of factors that stimulate intimal smooth muscle proliferation. The present studies indicate that the effects of chronic hyperlipidemia are complex in that the condition results not only in the deposition of lipids in the atheromatous lesions but that it may produce the primary endothelial injury that initiates the process of atherosclerosis as well.

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